Boronic Acid Salts Useful in Parenteral Formulations

ABSTRACT

Salts of a peptide boronic acid drug, for example of Cbz-(R)-Phe-(S)—Pro-(R)-Mpg-B(OH) 2 . The counter-ion to the boronate may be an alkali metal or derived from a strongly basic organic nitrogen-containing compound.

BACKGROUND

The present disclosure relates to pharmaceutically useful productsobtainable from organoboronic acids. The disclosure also relates to theuse of members of the aforesaid class of products, to their formulation,their preparation, their synthetic intermediates and to other subjectmatter.

The disclosure further relates to parenteral pharmaceutical formulationscontaining the described products.

Boronic Acid Compounds

It has been known for some years that boronic acid compounds and theirderivatives, e.g. esters, have biological activities, notably asinhibitors or substrates of proteases. For example, Koehler et al.Biochemistry 10:2477, 1971 report that 2-phenylethane boronic acidinhibits the serine protease chymotrypsin at millimolar levels. Theinhibition of chymotrypsin and subtilisin by arylboronic acids(phenylboronic acid, m-nitro-phenylboronic acid, m-aminophenylboronicacid, m-bromophenylboronic acid) is reported by Phillip et al, Proc.Nat. Acad. Sci. USA 68:478-480, 1971. A study of the inhibition ofsubtilisin Carlsberg by a variety of boronic adds, especially phenylboronic acids substituted by Cl, Br, CH₃, H₂N, MeO and others, isdescribed by Seufer-Wasserthal et al, Biorg. Med. Chem. 2(1):35-48,1994.

In describing inhibitors or substrates of proteases, P1, P2, P3, etc.designate substrate or inhibitor residues which are amino-terminal tothe scissile peptide bond, and S1, S2, S3, etc., designate thecorresponding subsites of the cognate protease in accordance with:Schechter, I. and Berger, A. On the Size of the Active Site inProteases, Biocherm. Biophys. Res. Comm., 27:157-162, 1967. In thrombin,the SI binding site or “specificity pocket” is a well defined slit inthe enzyme, whilst the 52 and S3 binding subsites (also respectivelycalled the proximal and distal hydrophobic pockets) are hydrophobic andinteract strongly with, respectively, Pro and (R)-Phe, amongst others.

Pharmaceutical research into serine protease inhibitors has moved fromthe simple arylboronic acids to boropeptides, i.e. peptides containing aboronic acid analogue of an α-amino carboxylic acid. The boronic acidmay be derivatised, often to form an ester. Shenvi (EP-A-145441 and U.S.Pat. No. 4,499,082) disclosed that peptides containing an α-aminoboronicacid with a neutral side chain were effective inhibitors of elastase andhas been followed by numerous patent publications relating toboropeptide inhibitors of serine proteases. Specific, tight bindingboronic acid inhibitors have been reported for elastase (K_(i), 0.25nM), chymotrypsin (K_(i), 0.25 nM), cathepsin G (K_(i), 21 nM), α-lyticprotease (K_(i), 0.25 nM), dipeptidyl aminopeptidase type IV (K_(i), 16pM) and more recently thrombin (Ac-D-Phe-Pro-boroArg-OH (DuP 714 initialK_(i) 1.2 nM).

Claeson et al (U.S. Pat. No. 5,574,014 and others) and Kakkar et al (WO92/07869 and family members Including U.S. Pat. No. 5,648,338) disclosethrombin inhibitors having a neutral C-terminal side chain, for examplean alkyl or alkoxyalkyl side chain.

Modifications of the compounds described by Kakkar et al are included inWO 96/25427, directed to peptidyl serine protease inhibitors In whichthe P2-P1 natural peptide linkage is replaced by another linkage. Asexamples of non-natural peptide linkages may be mentioned —CO₂—, —CH₂O—,—NHCO—, —CHYCH₂—, —CH═CH—, —CO(CH₂)_(p)CO— where p is 1, 2 or 3,—COCHY—, —CO₂—CH₂NH—, —CHY—NX—, —N(X) CH₂—N(X) CO—, —CH═C(CN)CO—,—CH(OH)—NH—, —CH(CN)—NH—, —CH(OH)CH₂— or —NH—CHOH—, where X is H or anamino protecting group and Y is H or halogen, especially F. Particularnon-natural peptide linkages are —CO₂— or —CH₂O—.

Metternich (EP 471651 and U.S. Pat. No. 5,288,707, the latter beingassigned to Trigen Limited) discloses variants of Phe-Pro-BoroArgboropeptides in which the P3 Phe is replaced by an unnatural hydrophobicamino acid such as trimethylsilylalanine,p-tert.butyl-diphenyl-silyloxymethyl-phenylalanine orp-hydroxymethylphenylalanine and the P1 side chain may be neutral(alkoxyalkyl, alkylthioalkyl or trimethylsilylalkyl).

The replacement of the P2 Pro residue of borotripeptide thrombininhibitors by an N-substituted glycine is described in Fevig J M et alBioorg. Med. Chem. 8: 301-306 and Rupin A et al Thromb. Haemost.78(4):1221-1227, 1997. See also U.S. Pat. No. 5,585,360 (de Nanteuil etal).

Amparo (WO 96/20698 and family members including U.S. Pat. No.5,698,538) discloses peptidomimetics of the structureAryl-linker-Boro(Aa), where Boro(Aa) may be an aminoboronate residuewith a non-basic side chain, for example BoroMpg. The linker is of theformula —(CH₂)_(m)CONR— (where m is 0 to 8 and R is H or certain organicgroups) or analogues thereof. In which the peptide linkage —CONR— isreplaced by —CSNR—, —SO₂NR—, —CO₂—, —C(S)O— or —SO₂O—. Aryl is phenyl,naphthyl or biphenyl substituted by one, two or three moieties selectedfrom a specified group. Most typically these compounds are of thestructure Aryl-(CH₂)_(n)—CONH—CHR²—BY¹Y², where R² is for example aneutral side chain as described above and n is 0 or 1.

Non-peptide boronates have been proposed as inhibitors of proteolyticenzymes in detergent compositions. WO 92/19707 and WO 95/12655 reportthat arylboronates can be used as Inhibitors of proteolytic enzymes indetergent compositions. WO 92/19707 discloses compounds substituted metato the boronate group by a hydrogen bonding group, especially acetamido(—NHCOCH₃), sulfonamido (—NHSO₂CH₃) and alkylamino. WO 95/12655 teachesthat ortho-substituted compounds are superior.

Boronate enzyme inhibitors have wide application, from detergents tobacterial sporulation inhibitors to pharmaceuticals. In thepharmaceutical field, there is patent literature describing boronateinhibitors of serine proteases, for example thrombin, factor Xa,kallikrein, elastase, plasmin as well as other serine proteases likeprolyl endopeptidase and Ig AI Protease. Thrombin is the last proteasein the coagulation pathway and acts to hydrolyse four small peptidesform each molecule of fibrinogen, thus deprotecting its polymerisationsites. Once formed, the linear fibrin polymers may be cross-linked byfactor XIIIa, which is Itself activated by thrombin. In addition,thrombin is a potent activator of platelets, upon which it acts atspecific receptors. Thrombin also potentiates its own production by theactivation of factors V and VIII.

Other aminoboronate or peptidoboronate inhibitors or substrates ofserine proteases are described in:

-   -   U.S. Pat. No. 4,935,493    -   EP 341661    -   WO 94/25049    -   WO 95/09859    -   WO 96/12499    -   WO 96/20689    -   Lee S -L et al, Biochemistry 36:13180-13186, 1997    -   Dominguez C et al, Bioorg. Med. Chem. Lett. 7:79-84, 1997    -   EP 471651    -   WO 94/20526    -   WO 95/20603    -   WO97/05161    -   U.S. Pat. No. 4,450,105    -   U.S. Pat. No. 5,106,948    -   U.S. Pat. No. 5,169,841.

Peptide boronic acid inhibitors of hepatic C virus protease aredescribed in WO 01/02424.

Matteson D S Chem. Rev. 89: 1535-1551, 1989 reviews the use of α-haloboronic esters as intermediates for the synthesis of Inter alia aminoboronic acids and their derivatives. Matteson describes the use ofpinacol boronic esters in non-chiral synthesis and the use of pinanedlolboronic esters for chiral control, including in the synthesis of aminoand amido boronate esters.

Contreras et al J. Organomet. Chem. 246: 213-217, 1983 describe howintramolecular N→B coordination was demonstrated by spectroscopicstudies on cyclic boronic esters prepared by reacting Me₂CHCMe₂-BH₂ withdiethanolamines.

Boronic acid and ester compounds have displayed promise as inhibitors ofthe proteasome, a multicatalytic protease responsible for the majorityof intracellular protein turnover. Ciechanover, Cell, 79:13-21, 1994,teaches that the proteasome is the proteolytic component of theubiquitin-proteasome pathway, in which proteins are targeted fordegradation by conjugation to multiple molecules of ubiquitin.Clechanover also teaches that the ubiquitin-proteasome pathway plays akey role in a variety of important physiological processes.

Adams et al, U.S. Pat. No. 5,780,454 (1998), U.S. Pat. No. 6,066,730(2000), U.S. Pat. No. 6,083,903 (2000) and equivalent WO 96/13266, andU.S. Pat. No. 6,297,217 (2001) describe peptide boronic ester and acidcompounds useful as proteasome inhibitors. These documents also describethe use of boronic ester and acid compounds to reduce the rate of muscleprotein degradation, to reduce the activity of NF-κB in a cell, toreduce the rate of degradation of p53 protein in a cell, to inhibitcyclin degradation in a cell, to Inhibit the growth of a cancer cell, toinhibit antigen presentation in a cell, to inhibit NF-κB dependent celladhesion, and to inhibit HIV replication. Brand et al, WO 98/35691,teaches that proteasome Inhibitors, including boronic acid compounds,are useful for treating infarcts such as occur during stroke ormyocardial infarction. Elliott et al, WO 99/15183, teaches thatproteasome inhibitors are useful for treating inflammatory andautoimmune diseases.

Unfortunately, organoboronic acids can be relatively difficult to obtainin analytically pure form. Thus, alkylboronic acids and their boroxinesare often air-sensitive. Korcek et al, J. Chem. Soc. Perkin Trans.2:242, 1972, teaches that butylboronic acid is readily oxidized by airto generate 1-butanol and boric acid.

It is known that derivatisation of boronic acids as cyclic estersprovides oxidation resistance. For example, Martichonok V et al J. Am.Chem. Soc. 118: 950-958, 1996 state that diethanolamine derivatisationprovides protection against possible boronic acid oxidation. U.S. Pat.No. 5,681,978 (Matteson D S et al) teaches that 1,2-diols and 1,3 diols,for example pinacol, form stable cyclic boronic esters that are noteasily oxidised.

Wu et al, J. Pharm. Sci., 89:758-765, 2000, discuss the stability of thecompound N-(2-pytazine) carbonyl-phenylalanine-leucine boronic acid(LDP-341, also known as bortezomib), an anti-cancer agent. It isdescribed how “during an effort to formulate [LDP-341] for parenteraladministration, the compound showed erratic stability behaviour”. Thedegradation pathways were Investigated and it was concluded that thedegradation was oxidative, the initial oxidation being attributed toperoxides or molecular oxygen and its radicals.

WO 02/059131 discloses botonic acid products which are described asstable. In particular, these products are certain boropeptides and/orboropeptidomimetics in which the boronic add group has been derivatisedwith a sugar. The disclosed sugar derivatives, which have hydrophobicamino acid side chains, are of the formula

wherein:

-   -   P is hydrogen or an amino-group protecting moiety;    -   R is hydrogen or alkyl;    -   A is 0, 1 or 2;    -   R¹, R² and R³ are independently hydrogen, alkyl, cycloalkyl,        aryl or —CH₂—R⁵;    -   R⁵, in each instance, is one of aryl, aralkyl, alkaryl,        cycloalkyl, heterocyclyl, heteroaryl, or —W—R⁶, where W is a        chalcogen and R⁶ is alkyl;    -   where the ring portion of any of said aryl, aralkyl, alkaryl,        cycloalkyl, heterocyclyl, or heteroaryl in R¹, R², R³ or R⁵ can        be optionally substituted; and    -   Z¹ and Z² together form a moiety derived from a sugar, wherein        the atom attached to boron in each case is an oxygen atom.

Some of the disclosed compounds are sugar derivatives of LDP-341 (seeabove).

Many drugs comprise an active moiety which is a carboxylic acid. Thereare a number of differences between carboxylic acids and boronic acids,whose effects on drug delivery, stability and transport (amongst others)have not been investigated. One feature of trivalent boron compounds isthat the boron atom is sp² hybridised, which leaves an empty 2p_(z)orbital on the boron atom. A molecule of the type BX₃ can therefore actas an electron-pair acceptor, or Lewis acid. It can use the empty 2p_(z)orbital to pick up a pair of nonbonding electrons from a Lewis base toform a covalent bond. BF₃ therefore reacts with Lewis bases such as NH₃to form acid-base complexes in which all of the atoms have a filledshell of valence electrons.

Boric acid, accordingly, can act as a Lewis acid, accepting OH—:

B(OH)₃+H₂O→B(OH)₄ ⁻+H⁺

Further, boronic adds of the type RB(OH)₂ are dibasic and have twopKa's. Another point of distinction about boron compounds is theunusually short length of bonds to boron, for which three factors may beresponsible:

1. Formation of pπ-pπ bonds;2. Ionic-covalent resonance;3. Reduced repulsions between non-bonding electrons.

The presumed equilibria of boronic and carboxylic adds in aqueous KOHare shown below (excluding formation of RBO₂ ²⁻):

Thrombosis

Hemostasis is the normal physiological condition of blood in which itscomponents exist in dynamic equilibrium. When the equilibrium isdisturbed, for instance following injury to a blood vessel, certainbiochemical pathways are triggered leading, in this example, to arrestof bleeding via clot formation (coagulation). Coagulation is a dynamicand complex process in which proteolytic enzymes such as thrombin play akey role. Blood coagulation may occur through either of two cascades ofzymogen activations, the extrinsic and intrinsic pathways of thecoagulation cascade. Factor VIIa in the extrinsic pathway, and FactorIXa in the intrinsic pathway are important determinants of theactivation of factor X to factor Xa, which itself catalyzes theactivation of prothrombin to thrombin, whilst thrombin in turn catalysesthe polymerization of fibrinogen monomers to fibrin polymer. The lastprotease in each pathway is therefore thrombin, which acts to hydrolyzefour small peptides (two FpA and two FpB) from each molecule offibrinogen, thus deprotecting its polymerization sites. Once formed, thelinear fibrin polymers may be cross-linked by factor XIIIa, which isitself activated by thrombin. In addition, thrombin is a potentactivator of platelets, upon which it acts at specific receptors.Thrombin activation of platelets leads to aggregation of the cells andsecretion of additional factors that further accelerate the creation ofa hemostatic plug, Thrombin also potentiates its own production by theactivation of factors V and VIII (see Hemker and Beguin in: Joiles, et.al., “Biology and Pathology of Platelet Vessel Wall Interactions,” pp.219-26 (1986), Crawford and Scrutton in: Bloom and Thomas, “Haemostasisand Thrombosis,” pp. 47-77, (1987), Bevers, et. al., Eur. J. Biochem.122:429-36, 1982, Mann, Trends Biochem. Sci. 12:229-33, 1987).

Proteases are enzymes which cleave proteins at specific peptide bonds.Cuypers et al., J. Biol. Chem. 257:7086, 1982, and the references citedtherein, classify proteases on a mechanistic basis into five classes:serine, cysteinyl or thiol, acid or aspartyl, threonine andmetalloproteases. Members of each class catalyse the hydrolysis ofpeptide bonds by a similar mechanism, have similar active site aminoacid residues and are susceptible to class-specific inhibitors. Forexample, all serine proteases that have been characterised have anactive site serine residue.

The coagulation proteases thrombin, factor Xa, factor VIIa, and factorIXa are serine proteases having trypsin-like specificity for the deavageof sequence-specific Arg-Xxx peptide bonds. As with other serineproteases, the cleavage event begins with an attack of the active siteserine on the scissile bond of the substrate, resulting in the formationof a tetrahedral intermediate. This is followed by collapse of thetetrahedral intermediate to form an acyl enzyme and release of the aminoterminus of the cleaved sequence. Hydrolysis of the acyl enzyme thenreleases the carboxy terminus.

As indicated above, platelets play two important roles in normalhemostasis. First, by aggregating, they constitute the initialhemostatic plug which immediately curtails bleeding from broken bloodvessels. Secondly, the platelet surface can become activated andpotentiate blood clotting, a property referred to as plateletprocoagulant activity. This may be observed as an increase in the rateof activation of prothrombin by factor Xa in the presence of factor Vaand Ca²⁺, referred to as the prothrombinase reaction. Normally, thereare few (If any) clotting factors on the surface of unstimulatedplatelets but, when platelets are activated, negatively chargedphospholipids (phosphatidylserine and phospatidylinositol) that arenormally on the cytoplasmic side of the membrane become available andprovide a surface on which two steps of the coagulation sequence occur.The phospholipid on the surface of activated platelets profoundlyaccelerates the reactions leading to the formation of thrombin, so thatthrombin can be generated at a rate faster than its neutralisation byantithrombin III. The reactions that occur on the platelet surfaces arenot easily inhibited by the natural anticoagulants in blood such asantithrombin III, either with or without heparin. (See Kelton and Hirschin: Bloom and Thomas, “Haemostasis and Thrombosis,” pp. 737-760, (1981);Mustard et al in: Bloom and Thomas, “Haemostasis and Thrombosis,” pp.503526, (1981); Goodwin et al; Biochem. J. 308:15-21, 1995).

A thrombus can be considered as an abnormal product of a normalmechanism and can be defined as a mass or deposit formed from bloodconstituents on a surface of the cardiovascular system, for example ofthe heart or a blood vessel. Thrombosis can be regarded as thepathological condition wherein improper activity of the hemostaticmechanism results in intravascular thrombus formation. Three basic typesof thrombi are recognised:

-   -   the white thrombus which is usually seen in arteries and        consists chiefly of platelets;    -   the red thrombus which is found in veins and is composed        predominantly of fibrin and red cells;    -   the mixed thrombus which is composed of components of both white        and red thrombi.

The composition of thrombi is influenced by the velocity of blood flowat their sites of formation. In general white platelet-rich thrombi formin high flow systems, while red coagulation thrombi form in regions ofstasis. The high shear rate in arteries prevents the accumulation ofcoagulation intermediates on the arterial side of the circulation: onlyplatelets have the capacity to form thrombi binding to the area ofdamage via von Willebrand factor. Such thrombi composed only ofplatelets are not stable and disperse. If the stimulus is strong thenthe thrombi will form again and then disperse continually until thestimulus has diminished. For the thrombus to stabilise, fibrin mustform. In this respect, small amounts of thrombin can accumulate withinthe platelet thrombus and activate factor Va and stimulate the plateletprocoagulant activity. These two events lead to an overall increase inthe rate of activation of prothrombin by factor Xa of 300,000 fold.Fibrin deposition stabilises the platelet thrombus. Indirect thrombininhibitors, for example heparin, are not clinically effective atinhibiting stimulation of platelet procoagulant activity. Accordingly, atherapeutic agent which inhibits platelet procoagulant activity would beuseful for treating or preventing arterial thrombotic conditions.

On the venous side of circulation, the thrombus is comprised of fibrin:thrombin can accumulate because of the slower flow on the venous sideand platelets play only a minor role.

Thrombosis is thus not considered to be a single indication but, rather,is a class of indications embracing distinct sub-classes for whichdiffering therapeutic agents and/or protocols may be appropriate. Thus,regulatory authorities treat disorders such as, for example, deep veinthrombosis, cerebrovascular arterial thrombosis and pulmonary embolismas distinct indications for the purposes of licensing medicines. Twomain sub-classes of thrombosis are arterial thrombosis and venousthrombosis. Arterial thrombosis includes such specific disorders asacute coronary syndromes [for example acute myocardial infarction (heartattack, caused by thrombosis in a coronary artery)], cerebrovasculararterial thrombosis (stroke, caused by thrombosis in the cerebrovasculararterial system) and peripheral arterial thrombosis. Examples ofconditions caused by venous thrombosis are deep vein thrombosis andpulmonary embolism.

The management of thrombosis commonly involves the use of antiplateletdrugs (inhibitors of platelet aggregation) to control futurethrombogenesis and thrombolytic agents to lyse the newly formed clot,either or both such agents being used in conjunction or combination withanticoagulants. Anticoagulants are used also preventatively(prophylactically) in the treatment of patients thought susceptible tothrombosis.

Currently, two of the most effective classes of drugs in clinical use asanticoagulants are the heparins and the vitamin K antagonists. Theheparins are ill-defined mixtures of sulfated polysaccharides that bindto, and thus potentiate, the action of antithrombin III. AntithrornbinIII is a naturally occurring inhibitor of the activated clotting factorsIXa, Xa, XIa, thrombin and probably XIIa (see Jaques, Pharmacol. Rev.31:99-166, 1980). The vitamin K antagonists, of which warfarin is themost well-known example, act indirectly by Inhibiting the post-ribosomalcarboxylations of the vitamin K dependent coagulation factors II, VII,IX and X (see Hirsch, Semin. Thromb. Hemostasis 12:1-11, 1986). Whileeffective therapies for the treatment of thrombosis, heparins andvitamin K antagonists have the unfortunate side effects of bleeding,heparin-induced thrombocytopenia (in the case of heparin) and markedinterpatient variability, resulting in a small and unpredictabletherapeutic safety margin.

The use of direct acting inhibitors of thrombin and other serineprotease enzymes of the coagulation system is expected to alleviatethese problems. To that end, a wide variety of serine proteaseInhibitors have been tested, including boropeptides, i.e. peptidescontaining a boronic acid analogue of an c-amino acid. Whilst directacting boronic acid thrombin Inhibitors have been discussed earlier inthis specification, they are further described in the following section.

Neutral P1 Residue Boropeptide Thrombin Inhibitors

Claeson et al (U.S. Pat. No. 5,574,014 and others) and Kakkar et al (WO92/07869 and family members including U.S. Pat. No. 5,648,338) discloselipophilic thrombin Inhibitors having a neutral (uncharged) C-terminal(P1) side chain, for example an alkoxyalkyl side chain.

The Claeson et al and Kakkar et al patent families disclose boronateesters containing the amino acid sequence D-Phe-Pro-BoroMpg[(R)-Phe-Pro-BoroMpg], which are highly specific inhibitors of thrombin.Of these compounds may be mentioned in particularCbz-(R)-Phe-Pro-BoroMpg-OPinacol (also known as TRI 50b). Thecorresponding free boronic acid is known as TRI 50c. For furtherinformation relating to TRI 50b and related compounds, the reader isreferred to the following documents:

-   Elgendy S et al., in The Design of Synthetic Inhibitors of Thrombin,    Claeson G et al Eds, Advances in Experimental Medicine, 340:173-178,    1993.-   Claeson G et al, Biochem 1290:309-312, 1993-   Tapparelli C et al, J. Biol Chem, 268:4734-4741, 1993-   Claeson G, in The Design of Synthetic Inhibitors of Thormbin,    Claeson G et al Eds, Advances in Experimental Medicine, 340:83-91,    1993-   Phillip et al, in The Design of Synthetic Inhibitors of Thrombin,    Claeson G et al Eds, Advances in Experimental Medicine, 340:67-77,    1993-   Tapparelli C et al, Trends Pharmacol. Sci. 14:366-376, 1993-   Claeson G, Blood Coagulation and Fibrinolysis 5:411-436, 1994-   Elgendy et al, Tetrahedron 50:3803-3812, 1994-   Deadman J. et al, J. Enzyme Inhibition 9:29-41, 1995-   Deadman J. et al, J. Medicinal Chemistry 38: 1511-1522, 1995.

The tripeptide sequence of TRI 50b has three chiral centres. The Pheresidue is considered to be of (R)-configuration and the Pro residue ofnatural (S)-configuration, at least in compounds with commerciallyuseful inhibitor activity; the Mpg residue is believed to be of(R)-configuration in isomers with commercially useful inhibitoractivity. Thus, the active, or most active, TRI 50b stereoisomer isconsidered to be of R,S,R configuration and may be represented as:

Whilst indirect acting thrombin inhibitors have been found usefulparenterally for the treatment of patients susceptible to or sufferingfrom venous thrombosis, the same is not true of arterial thrombosis,because it would be necessary to raise the dosage used in the treatmentof venous thrombosis by many times in order to treat (prevent) arterialthrombosis. Such raised dosages typically cause bleeding, which makesindirect acting thrombin inhibitors unsuitable or less preferable fortreating arterial thrombosis. Heparin and Its low molecular weightderivatives are indirect thrombin inhibitors, and so are unsuitable totreat arterial thrombosis. Oral direct thrombin inhibitors are indevelopment for arterial indications but may have lower than desirabletherapeutic indices, i.e. may have higher than desirable levels ofbleeding at therapeutic doses.

Many organoboronic acid compounds may be classified as lipophilic orhydrophobic. Typically, such compounds include amongst others:

-   -   boropeptides of which all or a majority of the amino acids are        hydrophobic    -   boropeptides of which at least half of the amino acids are        hydrophobic and which have a hydrophobic N-terminal substituent        (amino protecting group)    -   non-peptides based on hydrophobic moieties.

BRIEF SUMMARY OF THE DISCLOSURE

It has been discovered that TRI 50b tends to hydrolyse. Thus in acidconditions, for example of an HPLC assay, TRI 50b is converted to theadd form with a short half life, which implies potential hydrolysis inparenteral preparations containing water into species, comprising thefree add and its corresponding boronate anions in equilibrium therewith,taught in the literature to be unstable to degradation via de-boronation(carbon-boron bond cleavage), by an oxidative pathway (see e.g. Wu etal, discussed above).

The instability of TRI 50b to hydrolysis also presents potentialdisadvantages in preparation of the compound and its formulation, aswell as in the storage of pharmaceutical formulations containing it.

TRI 50c suffers further from Instability, in that there is a problematictendency for the boropeptide moiety itself to degrade via de-boronatlon(carbon-boron bond deavage), such deboronation being taught by theliterature to be oxidative (eg Wu et al, discussed above). The level ofdegradation can be remarkably high.

The properties discussed above of TRI 50b and TRI 50c will riot berestricted to such compounds but will be shared by other boropeptideesters and acids, even if the properties of such other boropeptidesdiffer quantitatively.

The present disclosure provides a solution to the problem of boronatediol ester and especially TRI 50b instability which also provides thecorresponding boronic acid with resistance to deboronation.

The present disclosure is predicated on, amongst other things, thefinding that certain organoboronic acid products are indicated to be ofenhanced stability.

The benefits of the present disclosure include a solution to the problemof boronate diol ester and especially TRI 50b instability, that is tosay the presently disclosed products provide inter aliapharmacologically active compounds which are more stable than TRI 50band other comparable esters in the sense of stability to hydrolysis. Thedisclosure further includes a solution to the problem of organoboronicacid instability, that Is to say the presently disclosed productsprovide inter alia pharmacologically active compounds which are morestable to deboronation than TRI 50c. The stability provided within theframework of the disclosure is not absolute but is improved relative tothe comparator compounds. The benefits offered by the disclosure furtherinclude the provision of products which have an unexpected usefulness inparenteral formulations.

There is disclosed an amino boronic acid derivative which avoids thedisadvantages of pinacol esters The disclosure further includes apeptide boronic acid derivative which is indicated to be of enhancedstability. In particular, the disclosure includes amongst other subjectmatter boronic add derivatives which are of relative stability tohydrolysis and deboronatlon and are useful in parenteral formulationsfor inhibiting thrombin.

The disclosure concerns a pharmaceutically acceptable base addition saltof certain organoboronic acid drugs, specifically hydrophobicboropeptides (e.g di- or tri-peptides), and more specifically thrombininhibitors having a non-basic P1 group. As a class, such salts are notonly contrary to the direction of the prior art but additionally have animproved level of stability which cannot be explained or predicted ontire basis of known chemistry.

In one aspect, the disclosure relates to pharmaceutically acceptablebase addition salt s of boronic acids which have a neutral aminoboronicacid residue capable of binding to the thrombin SI subsite linkedthrough a peptide linkage to a hydrophobic moiety capable of binding tothe thrombin S2 and S3 subsites. In a first embodiment, there isdisclosed a parenteral pharmaceutical formulation that includes apharmaceutically acceptable base addition salt of a boronic acid of, forexample, formula (I):

wherein

Y comprises a hydrophobic moiety which, together with the aminoboronicacid residue

—NHCH(R⁹)—B(OH)₂, has affinity for the substrate binding site ofthrombin; and

R⁹ is a straight chain alkyl group interrupted by one or more etherlinkages (e.g. 1 or 2) and In which the total number of oxygen andcarbon atoms is 3, 4, 5 or 6 (e.g. 5) or R⁹ is —(CH₂)_(m)—W where m is2, 3, 4 or 5 (e.g. 4) and W is —OH or halogen (F, Cl, Br or I). R⁹ is analkoxyalkyl group in one subset of compounds, e.g. alkoxyalkylcontaining 4 carbon atoms.

Disclosed as certain examples are pharmaceutically acceptable baseaddition salt s of hydrophobic boronic acid inhibitors of thrombin. Suchinhibitors may contain hydrophobic amino acids, and this class of aminoacids includes those whose side chain is hydrocarbyl, hydrocarbylcontaining an in-chain oxygen and/or linked to the remainder of themolecule by an in-chain oxygen or heteroaryl, or any of the aforesaidgroups when substituted by hydroxy, halogen or trifluoromethyl.Representative hydrophobic side chains include alkyl, alkoxyalkyl,either of the aforesaid when substituted by at least one aryl orheteroaryl, aryl, heteroaryl, aryl substituted by at least one alkyl andheteroaryl substituted by at least one alkyl. Proline and other iminoacids which are ring-substituted by nothing or by one of the moietieslisted in the previous sentence are also hydrophobic.

Some hydrophobic side chains contain from 1 to 20 carbon atoms, e.g.non-cyclic moieties having 1, 2, 3 or 4 carbon atoms. Side chainscomprising a cyclic group typically but not necessarily contain from 5to 13 ring members and in many cases are phenyl or alkyl substituted byone or two phenyls.

Included are inhibitors which contain hydrophobic non-peptide moieties,which are typically based on moieties which may form a side chain of ahydrophobic amino acid, as described above.

Hydrophobic compounds may contain, for example, one amino group and/orone acid group (e.g. —COOH, —B(OH)₂). Generally, they do not containmultiple polar groups of any one type.

The disclosure comprises pharmaceutically acceptable base addition salts of hydrophobic boronic acid inhibitors of thrombin, and thereforeincludes such salts of peptide boronic acids which have a partitioncoefficient between 1-n-octanol and water expressed as log P of greaterthan 1.0 at physiological pH and 25° C. Some peptide boronic acidsuseful in the Invention have a partition coefficient of at least 1.5. Aclass of hydrophobic peptide boronic acids useful in the invention has apartition coefficient of no more than 5.

Some sub-classes of hydrophobic organoboronic acids are those describedby Formulae (I) and (III) below, under the heading “Detailed Descriptionof Several Examples”.

Also disclosed as another embodiment is a pharmaceutically acceptablebase addition salt of a peptide boronic acid of formula (II):

where:

X is a moiety bonded to the N-terminal amino group and may be H to formNH₂. The identity of X is not critical but may be a particular X moietydescribed above. In one example there may be mentionedbenzyloxycarbonyl.

aa¹ is an amino acid having a hydrocarbyl side chain containing no morethan 20 carbon atoms (e.g. up to 15 and optionally up to 13 C atoms) andcomprising at least one cyclic group having up to 13 carbon atoms. Incertain examples, the cyclic group(s) of aa¹ have/has 5 or 6 ringmembers. For instance, the cyclic group(s) of aa¹ may be aryl groups,particularly phenyl. Typically, there are one or two cyclic groups inthe aa¹ side chain. Certain side chains comprise, or consist of, methylsubstituted by one or two 5- or 6-membered rings.

More particularly, aa¹ is Phe, Dpa or a wholly or partially hydrogenatedanalogue thereof. The wholly hydrogenated analogues are Cha and Dcha.

aa² is an imino acid having from 4 to 6 ring members. Alternatively, aa²is Gly N-substituted by a C₃-C₁₃ hydrocarbyl group, e.g. a C₃-C₈hydrocarbyl group comprising a C₃-C₆ hydrocarbyl ring; the hydrocarbylgroup may be saturated, for example exemplary N-substituents arecyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. As a hydrocarbylgroup containing one or more unsaturated bonds may be mentioned phenyland methyl or ethyl substituted by phenyl, e.g. 2-phenylethyl, as wellas β,β-dialkylphenylethyl.

There is a debate in the literature as to whether boronates in aqueoussolution form the ‘trigonal’ B(OH)₂ or ‘tetrahedral’ B(OH)₃— boronspecies, but NMR evidence seems to indicate that at a pH below the firstpKa of the boronic acid the main boron species is the neutral B(OH)₂. Inthe duodenum the pH is likely to be between 6 and 7, so the trigonalspecies is likely to be predominant here. In any event, the symbol—B(OH)₂ includes tetrahedral as well as trigonal boron species, andthroughout this specification symbols indicating trigonal boron speciesembrace also tetrahedral species. The symbol may further Include boronicgroups in anhydride form.

The salts may be in the form of solvates, particularly hydrates.

The salts may comprise, or consist essentially of, acid salts in whichthe boronic acid is singly deprotonated. The disclosure thereforeincludes products having a metal/boronate stoichiometry consistent withthe boronate groups in the product predominantly (more than 50 mol %)carrying a single negative charge.

Parenteral formulations of the salts are also provided herein. Inparticular, there are provided parenteral formulations comprising thesalts in the solid phase, for example particulate salts forreconstitution as aqueous solutions prior to administration by injectionor infusion. Such reconstituted solutions are also included in thedisclosure.

According to a further aspect of the present disclosure, there isprovided a method of treatment of a condition where anti-thromboticactivity is required which method comprises parenteral administration ofa therapeutically effective amount of a pharmaceutically acceptable baseaddition salt of a boronic acid of formula (I) to a person sufferingfrom, or at risk of suffering from, such a condition.

As described further hereinafter, there are provided also haemodialysissolutions comprising a salt of the disclosure.

The salts described herein include products obtainable by (having thecharacteristics of a product obtained by) reaction of the boronic acidwith a strong base and the term “salt” herein is to be understoodaccordingly. The term “salt” in relation to the disclosed products,therefore, does not necessarily imply that the products contain discretecations and anions and is to be understood as embracing products whichare obtainable using a reaction of a boronic acid and a base. Thedisclosure embraces products which, to a greater or lesser extent, arein the form of a coordination compound. The disclosure thus providesalso products obtainable by (having the characteristics of a productobtained by) reaction of a boronic acid (I) with a strong base a well asthe therapeutic, including prophylactic, use of such products.

The present disclosure is not limited as to the method of preparation ofthe salts, provided that they contain a boronate species derived fromboronic acid (I) and a counter-ion. Such boronate species may beboronate anions in any equilibrium form thereof. The term “equilibriumform” refers to differing forms of the same compounds which may berepresented in an equilibrium equation (e.g. boronic acid in equilibriumwith a boronic anhydride and in equilibrium with different boronateions). Boronates in the solid phase may form anhydrides and thedisclosed boronate salts when in the solid phase may comprise boronateanhydrides, as a boronic equilibrium species. It is not required thatthe salts be prepared by reaction of a base containing the counter-ionand the boronic acid (I). Further, the disclosure includes salt productswhich might be regarded as indirectly prepared by such an acid/basereaction as well as salts obtainable by (having the characteristics ofproducts obtained by) such indirect preparation. As examples of possiblyindirect preparation may be mentioned processes in which, after initialrecovery of the salt, it is purified and/or treated to modify itsphysicochemical properties, for example to modify solid form or hydrateform, or both.

In some embodiments, the cations of the salts are monovalent.

In some embodiments the salts comprise anhydride species; in others theyare essentially free of anhydride species.

The salts may be in isolated form. The salts may have a purity, e.g. asdetermined by the method of Example 34, of at least about 90%, e.g. ofgreater than or equal to about 95%. In the case of pharmaceuticalformulations, such salt forms may be combined with pharmaceuticallyacceptable diluents, excipients or carriers.

The disclosure includes a method for preparing the salts from thecorresponding boronic add as an intermediate, as well as theintermediate boronic acid of Formula (I) and a method for preparing it.

Further aspects and embodiments of the disclosure are set forth in thefollowing description and claims. Also included as such are the saltsdescribed herein.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of the words, for example“comprising” and “comprises”, mean “including but not limited to”, andare not intended to (and do not) exclude other moieties, additives,components, integers or steps.

This patent application contains data indicating that the stability(resistance to deboronation) of organoboronic acids may be increased byproviding them in the form of salts, e.g. metal salts. In singleexperiments, the ammonium salt of TRI 50c appeared to decompose ondrying to yield ammonia, whilst the choline salt demonstrated rapiddecomposition to a deboronated impurity. Although experiments have notbeen conducted to reproduce these unrepeated observations, there isprovided a sub-class in which the ammonium and choline salts areexcluded. The salt may be an acid salt. In any event, this stabilisationtechnique forms part of the disclosure and is applicable, inter alia, toorganoboronic acids described under the heading “BACKGROUND” and toorganoboronic acids described in publications mentioned under thatheading.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart referred to in Example 35, showing the results of athrombin amidolytic assay of TRI 1405 (TRI 50c magnesium salt) and TRI50b, where Vmax is the maximum rate of reaction measured by amidolyticassay.

FIG. 2 is a plot referred to in Example 39, showing intravenous phaseclearance and kinetics following a single dose of TRI 50b or TRI 50c.

DETAILED DESCRIPTION OF SEVERAL EXAMPLES Glossary

The following terms and abbreviations are used in this specification:

The expression “acid salt” as applied to a salt of a boronic acid refersto salts of which a single —OH group of the trigonally-represented acidgroup —B(OH)₂ is deprotonated. Thus salts wherein the boronate groupcarries a single negative charge and may be represented as —B(OH)(O—) oras [—B(OH)₃]⁻are acid salts. The expression encompasses salts of acation having a valency n wherein the molar ratio of boronic acid tocation is approximately n to 1. In practical terms, the observedstoichiometry is unlikely to be exactly n:1, but will be consistent witha notional n:1 stoichiometry, For example, the observed mass of thecation might vary from the calculated mass for a n:1 stoichiametry by nomore than about 10%, e.g. no more than about 7.5%; in some cases anobserved mass of a cation might vary from the calculated mass by no morethan about 1%. Calculated masses are suitably based on the trigonal formof the boronate. (At an atomic level, a salt stoichiometricallyconsistent with being an acid salt might contain boronates in a mix ofprotonation states, whose average approximates to single deprotonationand such “mixed” salts are included in the term “add salt”). Examples ofacid salts are monosodium salts and hemicalcium salts.

α-Aminoboronic acid or Boro(aa) refers to an amino acid in which the CO₂group has been replaced by BO₂.

The term “amino-group protecting moiety” refers to any group used toderivatise an amino group, especially an N-terminal amino group of apeptide or amino acid. Such groups include, without limitation, alkyl,acyl, alkoxycarbonyl, aminocarbonyl, and sulfonyl moieties. However, theterm “amino-group protecting moiety” is not intended to be limited tothose particular protecting groups that are commonly employed in organicsynthesis, nor is it intended to be limited to groups that are readilycleavable.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings or animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The expression “thrombin inhibitor” refers to a product which, withinthe scope of sound pharmacological judgement, is potentially or actuallypharmaceutically useful as an inhibitor of thrombin, and includesreference to substance which comprises a pharmaceutically active speciesand is described, promoted or authorised as a thrombin inhibitor. Suchthrombin inhibitors may be selective, that is they are regarded, withinthe scope of sound pharmacological judgement, as selective towardsthrombin in contrast to other proteases; the term “selective thrombininhibitor” includes reference to substance which comprises apharmaceutically active species and is described, promoted or authorisedas a selective thrombin inhibitor.

The term “heteroaryl” refers to a ring system which has at least one(e.g. 1, 2 or 3) in-ring heteroatoms and has a conjugated in-ring doublebond system. The term “heteroatom” includes oxygen, sulfur and nitrogen,of which sulfur is sometimes less preferred.

“Natural amino acid” means an L-amino acid (or residue thereof) selectedfrom the following group of neutral (hydrophobic or polar), positivelycharged and negatively charged amino acids:

Hydrophobic Amino Acids

-   -   A=Ala=alanine    -   V=Val=valine    -   I=Ile=isoleucine    -   L=Leu=leucine    -   M=Met methionine    -   F Phe=phenylalanine    -   P Pro=proline    -   W=Trp=tryptophan

Polar (Neutral or Uncharged) Amino Acids

-   -   N=Asn=asparagine    -   C=Cys=cysteine    -   Q=Gln=glutamine    -   G=Gly=glycine    -   S=Ser=serine    -   T=Thr=threonine    -   Y=Tyr=tyrosine

Positively Charged (Basic) Amino Acids

-   -   R=Arg=arginine    -   H=His=histidine    -   K=Lys=lysine

Negatively Charged Amino Acids

-   -   D=Asp=aspartic add    -   E=Glu=glutamic add.        ACN=acetonitrile        Amino acid=α-amino acid        Base addition salt=a salt which is prepared from addition of an        inorganic base or an organic base to a free acid (In this case        the boronic add).        Cbz=benzyloxycarbonyl        Cha=cyclohexylalanine (a hydrophobic unnatural amino acid)        Charged (as applied to drugs or fragments of drug molecules,        e.g. amino acid residues)=carrying a charge at physiological pH,        as in the case of an amino, amidino or carboxy group        Dcha=dicyclohexylalanine (a hydrophobic unnatural amino acid)        Dpa=diphenylalanine (a hydrophobic unnatural amino acid)        Drug=a pharmaceutically useful substance, whether the active in        vivo principle or a prodrug        i.v.=intravenous        Mpg=3-methoxypropylglycine (a hydrophobic unnatural amino acid)        Multivalent=valency of at least two, for example two or three        Neutral (as applied to drugs or fragments of drug molecules,        e.g. amino acid residues)=uncharged        =not carrying a charge at physiological pH        Pinac=Pinacol=2,3-dimethyl-2,3-butanediol        Pinanedlol=2,3-pinanediol=2,6,6-trimethylbicyclo        [3.1.1]heptarie-2,3-diol        Pip=pipecolinic acid        s.c.=subcutaneous        Strong base=a base having a sufficiently high pKb to react with        a boronic acid. Suitably such bases have a pKb of 7 or more,        e.g. 7.5 or more, for example about 8 or more THF        tetrahydrofuran        Thr thrombin

The Compounds

The products of the disclosure comprise salts of boronic acids whichhave a neutral aminoboronic acid residue capable of binding to thethrombin S1 subsite linked through a peptide linkage to a hydrophobicmoiety capable of binding to the thrombin 52 and S3 subsites. Thedisclosure includes salts of acids of formula (I):

wherein

Y comprises a hydrophobic moiety which, together with the aminoboronicacid residue —NHCH(R⁹)—B(OH)₂, has affinity for the substrate bindingsite of thrombin; and

R⁹ is a straight chain alkyl group Interrupted by one or more etherlinkages and in which the total number of oxygen and carbon atoms is 3,4, 5 or 6 (e.g. 5) or R⁹ is —(CH₂)_(m)—W where m is from 2, 3, 4 or 5(e.g. 4) and W is —OH or halogen (F, Cl, Br or I). As examples ofstraight chain alkyl interrupted by one or more ether linkages (—O—) maybe mentioned alkoxyalkyl (one interruption) and alkoxyalkoxyalkyl (twointerruptions). R⁹ is an alkoxyalkyl group in one subset of compounds,e.g. alkoxyalkyl containing 4 carbon atoms.

Typically, YCO— comprises an amino acid residue (whether natural orunnatural) which binds to the S2 subsite of thrombin, the amino acidresidue being N-terminally linked to a moiety which binds the S3 subsiteof thrombin.

In one class of Formula (I) acids, YCO— is an optionally N-terminallyprotected dipeptide residue which binds to the 53 and S2 binding sitesof thrombin and the peptide linkages in the acid are optionally andindependently N-substituted by a C₁-C₁₃ hydrocarbyl group optionallycontaining in-chain and/or in-ring nitrogen, oxygen or sulfur andoptionally substituted by a substituent selected from halo, hydroxy andtrifluoromethyl. The N-terminal protecting group, when present, may be agroup X as defined above (other than hydrogen). Normally, the acidcontains no N-substituted peptide linkages; where there is anN-substituted peptide linkage, the substituent is often 1C to 6Chydrocarbyl, e.g. saturated hydrocarbyl; the N-substituent comprises aring in some embodiments, e.g. cycloalkyl, and may be cyclopentyl, forexample. One class of acids has an N-terminal protecting group (e.g. anX group) and unsubstituted peptide linkages.

Where YCO— is a dipeptide residue (whether or not N-terminallyprotected), the S3-binding amino acid residue may be of R configurationand/or the S2-binding residue may of S configuration. The fragment—NHCH(R⁹)—B(OH) may of R configuration. The disclosure is not restrictedto chiral centres of these conformations, however.

In one class of compounds, the side chain of P3 (S3-binding) amino acidand/or the P2 (S2-binding) amino acid is a moiety other than hydrogenselected from a group of formula A or B:

—(CO)_(a)—(CH₂)_(b)-D_(C)-(CH₂)_(d)-E  (A)

—(CO)_(a)—(CH₂)_(b)-D_(C)-C_(e)(E¹)(E²)(E³)  (B)

wherein

a is 0 or 1;e is 1;b and d are independently 0 or an integer such that (b+d) is from 0 to 4or, as the case may be, (b+e) is from 1 to 4;c is 0 or 1;

D is O or S;

E is H, C₁-C₆ alkyl, or a saturated or unsaturated cyclic group whichnormally contains up to 14 members and particularly is a 5-6 memberedring (e.g. phenyl) or an 8-14 membered fused ring system (e.g.naphthyl), which alkyl or cyclic group is optionally substituted by upto 3 groups (e.g. 1 group) independently selected from C₁-C₆trialkylsilyl, —CN, —R¹³, —R¹²OR¹³, —R¹²COR¹³, —R¹²CO₂R¹³ and —R²CR¹³,wherein R¹² is —(CH₂)_(f)— and R¹³ is —(CH₂)_(g)H or by a moiety whosenon-hydrogen atoms consist of carbon atoms and in-ring heteroatoms andnumber from 5 to 14 and which contains a ring system (e.g. an arylgroup) and optionally an alkyl and/or alkylene group, wherein f and gare each independently from 0 to 10, g particularly being at least 1(although —OH may also be mentioned as a substituent), provided that(f+g) does not exceed 10, more particularly does not exceed 6 and mostparticularly is 1, 2, 3 or 4, and provided that there is only a singlesubstituent if the substituent is a said moiety containing a ringsystem, or E is C₁-C₆ trialkylsilyl; and E¹, E² and E³ are eachindependently selected from —R¹⁵ and -J—R¹⁵, where J is a 5-6 memberedring and R¹⁵ is selected from C₁-C₆ trialkylsilyl, —CN, —R¹³, —R¹²OR¹³,—R¹²COR¹³, —R¹²CO₂R¹³, —R¹²O₂CR¹³, and one or two halogens (e.g. in thelatter case to form a -J-R¹⁵ moiety which is dichlorophenyl), where R¹²and R¹³ are, respectively, an R¹² moiety and an R¹³ moiety as definedabove (in some acids where E¹, E² and E³ contain an R¹³ group, g is 0 or1);

in which moiety of Formula (A) or (B) any ring is carbocyclic oraromatic, or both, and any one or more hydrogen atoms bonded to a carbonatom is optionally replaced by halogen, especially F.

In certain examples, a is 0. If a is 1, c may be 0. In particularexamples, (a+b+c+d) and (a+b+c+e) are no more than 4 and are moreespecially 1, 2 or 3. (a+b+c+d) may be 0.

Exemplary groups for E, E¹, E² and E³ include aromatic rings such asphenyl, naphthyl, pyridyl, quinolinyl and furanyl, for example;non-aromatic unsaturated rings, for example cyclohexenyl; saturatedrings such as cyclohexyl, for example. E may be a fused ring systemcontaining both aromatic and non-aromatic rings, for example fluorenyl.One class of E, E¹, E² and E³ groups are aromatic (includingheteroaromatic) rings, especially 6-membered aromatic rings. In somecompounds, E¹ is H whilst E² and E³ are not H; in those compounds,examples of E² and E³ groups are phenyl (substituted or unsubstituted)and C₁-C₄ alkyl, e.g. methyl.

In one class of embodiments, E contains a substituent which is C₁-C₆alkyl, (C₁-C₅ alkyl)carbonyl, carboxy C₁-C₅ alkyl, aryl (includingheteroaryl), especially 5-membered or preferably 6-membered aryl (e.g.phenyl or pyridyl), or arylalkyl (e.g. arylmethyl or arylethyl wherearyl may be heterocyclic and is preferably 6-membered)).

In another class of embodiments, E contains a substituent which is OR¹³,wherein R¹³ can be a 6-membered ring, which may be aromatic (e.g.phenyl) or is alkyl (e.g. methyl or ethyl) substituted by such a6-membered ring.

A class of moieties of formula A or B are those in which E is a6-membered aromatic ring optionally substituted, particularly at the2-position or 4-position, by —R¹³ or —OR¹³.

The disclosure includes salts in which the P3 and/or P2 side chaincomprises a cyclic group in which 1 or 2 hydrogens have been replaced byhalogen, e.g. F or Cf.

The disclosure includes a class of salts in which the side chains offormula (A) or (B) are of the following formulae (C), (D) or (E):

wherein q is from 0 to 5, e.g. is 0, 1 or 2, and each T is independentlyhydrogen, one or two halogens (e.g. F or Cl), —SiMe₃, —CN, —R¹³, —OR¹³,—COR¹³, —CO₂R¹³ or —O₂CR¹³. In some embodiments of structures (D) and(E), T is at the 4-position of the phenyl group(s) and is —R¹³, —OR¹³,—COR¹³, —CO₂R¹³ or —O₂CR¹³, and R¹³ is C₁-C₁₀ alkyl and moreparticularly C₁-C₆ alkyl. In one sub-class, T is —R¹³ or —OR¹³, forexample 1n which f and g are each independently 0, 1, 2 or 3;

In some side chains groups of this sub-class, T is —R¹²OR¹³ and R¹³ isH. In one class of the moieties, the side chain is of formula (C) andeach T is independently R¹³ or OR¹³ and R¹³ is C₁-C₄ alkyl. In some ofthese compounds, R¹³ is branched alkyl and in others it is straightchain. In some moieties, the number of carbon atoms is from 1 to 4.

In many dipeptide fragments YCO— (which dipeptides may be N-terminallyprotected or not), the P3 amino acid has a side chain of formula (A) or(B) as described above and the P2 residue is of an imino acid.

The disclosure therefore includes medicaments comprising salts, e.g.metal salts, of organoboronic acids which are thrombin inhibitors,particularly selective thrombin Inhibitors, having a neutral P1(S1-binding) moiety. For more information about moieties which bind tothe 53, 52 and SI sites of thrombin, see for example Tapparelli C et al,Trends Pharmacol. Sci. 14: 366-376, 1993; Sanderson P et al, CurrentMedicinal Chemistry, 5: 289-304, 1998; Rewinkel J et al, CurrentPharmaceutical Design, 5:1043-1075, 1999; and Coburn C Exp. Opin. Ther.Patents 11(5): 721-738, 2001. The thrombin inhibitory salts of thedisclosure are not limited to those having S3, S2 and SI affinity groupsdescribed in the publications listed in the preceding sentence.

The boronic acids may have a Ki for thrombin of about 100 nM or less,e.g. about 20 nM or less.

A subset of the Formula (I) acids comprises the acids of Formula (III):

X is a moiety bonded to the N-terminal amino group and may be H to formNH₂. The identity of X is not critical but may be a particular X moietydescribed above. In one example there may be mentionedbenzyloxycarbonyl.

In certain examples X is R⁶—(CH₂)_(p)—C(O)—, R⁶—(CH₂)_(p)—S(O)₂—,R⁶—(CH₂)_(p)—NH—C(O)— or R⁶—(CH₂)_(p)—O—C(O)— wherein p is 0, 1, 2, 3,4, 5 or 6 (of which 0 and 1 are preferred) and R⁶ is H or a 5 to13-membered cyclic group optionally substituted by 1, 2 or 3substituents selected from halogen, amino, nitro, hydroxy, a C₅-C₆cyclic group, C₁-C₄ alkyl and C₁-C₄ alkyl containing, and/or linked tothe 5 to 13-membered cyclic group through, an in-chain 0, the aforesaidalkyl groups optionally being substituted by a substituent selected fromhalogen, amino, nitro, hydroxy and a C₅-C₆ cyclic group. Moreparticularly X is R⁶—(CH₂)_(p)—C(O)— or R⁶—(CH₂)_(p)—O—C(O)— and p is 0or 1. Said 5 to 13-membered cyclic group is often aromatic orheteroaromatic, for example is a 6-membered aromatic or heteroaromaticgroup. In many cases, the group is not substituted.

Exemplary X groups are (2-pyrazine) carbonyl, (2-pyrazine) sulfonyl andparticularly benzyloxycarbonyl.

aa¹ is an amino acid residue having a hydrocarbyl side chain containingno more than 20 carbon atoms (e.g. up to 15 and optionally up to 13 Catoms) and comprising at least one cyclic group having up to 13 carbonatoms. In certain examples, the cyclic group(s) of aa¹ have/has 5 or 6ring members. For instance, the cyclic group(s) of aa¹ may be arylgroups, particularly phenyl. Typically, there are one or two cyclicgroups in the aa¹ side chain. Certain side chains comprise, or consistof, methyl substituted by one or two 5- or 6-membered rings.

More particularly, aa¹ is Phe, Dpa or a wholly or partially hydrogenatedanalogue thereof. The wholly hydrogenated analogues are Cha and Dcha.

aa² is an Imino add residue having from 4 to 6 ring members.Alternatively, aa² is Gly N-substituted by a C₃-C₁₃ hydrocarbyl group,e.g. a C₃-C₈ hydrocarbyl group comprising a C₃-C₆ hydrocarbyl ring; thehydrocarbyl group may be saturated, for example exemplary N-substituentsare cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. As ahydrocarbyl group containing one or more unsaturated bonds may bementioned phenyl and methyl or ethyl substituted by phenyl, e.g.2-phenylethyl, as well as β,β-dialkylphenylethyl.

An exemplary class of products comprises those in which aa² is a residueof an imino acid of formula (IV)

where R¹¹ is —CH₂—, CH₂—CH₂—, —S—CH₂— or CH₂—CH₂—CH₂—, which group whenthe ring is 5 or 6-membered is optionally substituted at one or more—CH₂— groups by from 1 to 3 C₁-C₃ alkyl groups, for example to form theR¹¹ group —S—C(CH₃)₂—. Of these Imino acids, azetidine-2-carboxylicacid, especially (s)-azetidine-2-carboxylic add, and more particularlyproline are illustrative.

It will be appreciated from the above that a very preferred class ofproducts consists of those in which aa¹-aa² is Phe-Pro. In anotherpreferred class, aa¹-aa² is Dpa-Pro. In other products, aa¹-aa² isCha-Pro or Dcha-Pro. Of course, also included are corresponding productclasses in which Pro is replaced by (s)-azetidine-2-carboxylic acid.

R⁹ is as defined previously and may be a moiety R¹ of the formula—(CH₂)_(s)-Z. Integer s is 2, 3 or 4 and W is —OH, —OMe, —OEt or halogen(F, Cl, I or, preferably, Br). Particularly illustrative Z groups are—OMe and —OEt, especially —OMe. In certain examples s is 3 for all Zgroups and, Indeed, for all compounds of the disclosure. Particular R¹groups are 2-bromoethyl, 2-chloroethyl, 2-methoxyethyl, 4-bromobutyl,4-chlorobutyl, 4-methoxybutyl and, especially, 3-bromopropyl,3-chloropropyl and 3-methoxypropyl. Most preferably, R¹ is3-methoxypropyl. 2-Ethoxyethyl is another preferred R¹ group.

Accordingly, a specific class of salts consists of those of acids of theformula X-Phe-Pro-Mpg-B(OH)₂, especially Cbz-Phe-Pro-Mpg-B(OH)₂; alsoincluded are analogues of these compounds in which Mpg is replaced by aresidue with another of the R¹ groups and/or Phe is replaced by Dpa oranother aa¹ residue.

The aa¹ moiety of the salt is preferably of (R)-configuration. The aa²moiety is preferably of (S)-configuration. Particularly preferred saltshave aa¹ of (R)-configuration and aa² of (S)-configuration. The chiralcentre —NH—CH(R¹)—B— is preferably of (R)-configuration. It isconsidered that commercial formulations will have the chiral centres in(R,S,R) arrangement, as for example in the case of salts ofCbz-Phe-Pro-BoroMpg-OH:

The disclosure includes salts of Cbz-(R)-Phe-(S)—Pro-(R)-boroMpg-OH (andof other compounds of the formula X—(R)-Phe-(S)Pro-(R)-boroMpg-OH) whichare at least 90% pure, e.g. at least 95% pure.

In broad terms, the salts described herein may be considered tocorrespond to reaction products of an organoboronic acid as describedabove with a strong base, e.g. a basic metal compound; the salts arehowever not limited to products resulting from such a reaction and maybe obtained by alternative routes.

The salts are therefore obtainable by contacting an acid of formula (I)with a strong base. The disclosure thus contemplates products(compositions of matter) having the characteristics of a reactionproduct of an acid of formula (I) and a strong base. The base ispharmaceutically acceptable.

As suitable salts may be mentioned salts of metals, e.g. of monovalentor divalent metals, and stronger organic bases, for example:

1. Alkali metal salts;2. Divalent, eg alkaline earth metal, salts;3. Group III metals;4. Salts of strongly basic organic nitrogen-containing compounds,including:

-   -   4A. Salts of guanidines and their analogues;    -   4B. Salts of strongly basic amine, examples of which include (i)        aminosugars and (ii) other amines.

Of the above salts, particularly illustrative are alkali metals,especially Na and Li. Also Illustrative are aminosugars.

Specific salts are of the acid boronate though in practice the add saltsmay contain a very small proportion of the doubly deprotonated boronate.The term “acid boronate” refers to trigonal —B(OH)₂ groups in which oneof the B—OH groups is deprotonated as well as to correspondingtetrahedral groups in equilibrium therewith. Acid boronates have astoichiometry consistent with single deprotonation.

The disclosure includes therefore products (compositions of matter)which comprise salts which may be represented by formula (V):

where Y^(n+) is a pharmaceutically acceptable cation obtainable from astrong base, and aa¹, aa², X and R¹ are as defined above. Also includedare products in which R¹ is replaced by another R⁹ group.

One class of salts have a solubility of about 10 mM or more, e.g. of atleast about 20 mM, when their solubility is determined as described inthe examples at a dissolution of 25 mg/ml. More particularly yet theyhave a solubility of least 50 mM when their solubility is determined asdescribed in the examples at a dissolution of 50 mg/ml.

The disclosure includes salts of boronic acids (I) having an observedstoichiometry consistent with the salt being of (being representable by)the formula “(boronate⁻)_(n) cation^(n+)”. One class of such salts arerepresented by the formula:

[Cbz-(R)-Phe-(S)—Pro-(R)-Mpg-B(OH)(O⁻)]M⁺

where M⁺ represents a monovalent cation, especially an alkali metalcation. It will be understood that the above representation is anotional representation of a product whose observed stoichiometry isunlikely to be literally and exactly 1:1. In any event, a particularsalt is Cbz-(R)-Phe-(S)—Pro-(R)-Mpg-B(OH)₂ monosodium salt (TGN 255). Inthe above formula, the trigonally-represented boronate represents, asalways, boronates which are trigonal, tetrahedral or mixedtrigonal/tetrahedral.

Particularly Exemplary are Products which Comprise:(i) species selected from (a) acids of formula (VIII):X—(R)-Phe-(S)—Pro-(R)-Mpg-B(OH)₂ where X is H or an amino-protectinggroup, especially Cbz, (b) boronate anions thereof, and (c) anyequilibrium form of the aforegoing (e.g. an anhydride); and(ii) Ions having a valency n in combination with said species, thespecies and said ions having an observed stoichiometry consistent with anotional species:ion stoichiometry of n:1. In one class of salts, n is1.

Considering the Counter-Ions in Turn: 1. Monovalent Metal, EspeciallyAlkali Metal Salts

Suitable alkali metals include lithium, sodium and potassium. All ofthese are remarkably soluble. Lithium and sodium are illustrativebecause of their high solubility. The lithium and particularly sodiumsalts are of surprisingly high solubility in relation to potassiumamongst others. Sodium is most used in many instances. Salts containingmixtures of alkali metals are contemplated by the disclosure.

The disclosure includes products comprising salts of the formula (VI)

where M⁺ is an alkali metal ion and aa¹, aa², X and R¹ are as definedabove, as well as salts in which both hydroxy groups of the boronategroup are in salt form (preferably with another identical M⁺ group) andmixtures of such salts. Included also are products wherein R¹ isreplaced by another R⁹ group.

2. Divalent. e.g. Alkaline Earth Metal (Group II Metal) Salts

One example of a divalent metal is calcium. Another suitable divalentmetal is magnesium. Also contemplated is zinc. The divalent metals areusually used in a boronic acid:metal ratio of substantially 2:1, inorder to achieve the preferred monovalent boronate moiety. Saltscontaining mixtures of divalent metals, e.g. mixtures of alkaline earthmetals, are also contemplated.

Further disclosed are products (compositions of matter) which comprisesalts which may be represented by the formula (VII):

where M²⁺ is a divalent metal cation, e.g. an alkaline earth metal orzinc cation, and aa¹, aa², X and R⁹ are as defined above, as well assalts in which both hydroxy groups of the boronate group aredeprotonated and mixtures of such salts. As previously indicated, theboronate may comprise a tetrahedral species.

3. Group III Metals

Suitable Group III metals include aluminium and gallium, Saltscontaining mixtures of Group III metals are also contemplated.

The disclosure includes products comprising salts of the formula (VIII):

where M³⁺ is a Group III metal ion and aa¹, aa², X and R⁹ are as definedabove, as well as salts in which both hydroxy groups of the boronategroup are in salt form and mixtures of such salts. As previouslyindicated, the boronate may comprise a tetrahedral species.

4. Strongly Basic Organic Nitrogen-Containing Compounds

The disclosure includes products obtainable by (having thecharacteristics of a product obtained by) reaction of a peptide boronicacid as defined above and a strong organic base. Two illustrativeclasses of organic base are described in sections 4A and 4B below.Particularly preferred are acid salts (in which one of the two boronic—OH groups is deprotonated). Most commonly, the salts contain a singletype of organic counter-ion (disregarding trace contaminants) but thedisclosure contemplates salts containing mixtures of organiccounter-ions; in one sub-class, the different counter-ions all fallwithin the section 4A family described below or, as the case may be, inthe section 4B family below; in another subclass, the salts comprise amixture of organic counter-ions which are not all from the same family(4A or 4B).

Suitable organic bases include those with a pKb of 7 or more, e.g. 7.5or more, for example in the region of 8 or more. Bases which are lesslipophilic [e.g. have at least one polar functional group (e.g. 1, 2 or3 such groups) for example hydroxy] are favoured; thus aminosugars areone favoured class of base.

4A. Guanidines and their Analogues

The guanidino compound (guanidine) may in principle be any soluble andpharmaceutically acceptable compound having a guanidino or a substitutedguanidino group, or a substituted or unsubstituted guanidine analogue.Suitable substituents include aryl (e.g. phenyl), alkyl or alkylinterrupted by an ether or thioether linkage and, in any event,typically contain from 1 to 6 and especially 1, 2, 3, or 4 carbon atoms,as in the case of methyl or ethyl. The guanidino group may have 1, 2, 3or 4 substituent groups but more usually has 1 or 2 substituent groups,for instance on a terminal nitrogen. One class of guanidines ismonoalkylated; another class is dialkylated. As guanidine analogues maybe mentioned thioguanidines and 2-amino pyridines. Compounds havingunsubstituted guanidino groups, for example guanidine and arginine, formone particular class.

Salts containing mixtures of guanidines are contemplated by thedisclosure.

A particular guanidino compound is L-arginine or an L-arginine analogue,for example D-arginine, or the D- or, preferably, L-isomers ofhomoarginine or agmatine [(4-aminobutyl) guanidine]. Less preferredarginine analogues are NG-nitro-L-arginine methyl ester, for example,and constrained guanidine analogues, particularly 2-amino pyrimidines,for example 2,6-quinazolinediamines such as5,6,7,8-tetrahydro-2,6-quinazolinediamine, for example. The guanidinocompound may also be a peptide, for example a dipeptide, containingarginine; one such dipeptide is L-tyrosyl-L-arginine.

Some particular guanidino compounds are compounds of formula (VII):

where n is from 1 to 6 and for example at least 2, e.g. 3 or more, andin many instances no more than 5. Most particularly, n is 3, 4 or 5, R²is H or carboxylate or derivatised carboxylate, for example to form anester (e.g. a C₁-C₄ alkyl ester) or amide. R³ is H, C₁-C₄ alkyl or aresidue of a natural or unnatural amino acid (e.g, tyrosine). Thecompounds of formula (IV) are usually of L-configuration. The compoundsof formula (IV) are arginine (n=3; R²=carboxyl; R³═H) and argininederivatives or analogues.

The disclosure includes products comprising salts of the formula (IX)

where aa¹, aa², X and R¹ are as defined previously and G⁺ is theprotonated form of a pharmaceutically acceptable organic compoundcomprising a guanidino group or an analogue thereof, as well as salts inwhich both hydroxy groups of the boronate group are in salt form(preferably with another Identical G+group) and mixtures of such salts.Also included are products wherein R¹ is replaced by another R⁹ group.

4B. Strongly Basic Amines

The disclosure includes products obtainable by (having thecharacteristics of a product obtained by) reaction of a peptide boronicacid as defined above and a strong organic base which is an amine. Theamine may in principle be any soluble and pharmaceutically acceptableamine.

It is envisaged that a desirable class of amine includes those havingpolar functional groups in addition to a single amine group, as suchcompounds will be more hydrophilic and thus more soluble than others. Incertain salts, the or each additional functional group is hydroxy. Someamines have 1, 2, 3, 4, 5 or 6 additional functional groups, especiallyhydroxy groups. In one Illustrative class of amines the ratio of (aminoplus hydroxy groups):carbon atoms is from 1:2 to 1:1, the latter ratiobeing particularly preferred. These amines with one or more additionalpolar functional groups may be a hydrocarbon, especially an alkane,substituted by the amino group and the additional polar group(s). Theamino group may be substituted or unsubstituted and, excluding aminosubstituents, the polar base may contain, for example, up to 10 carbonatoms; usually there are no less than three such carbon atoms, e.g. 4, 5or 6. Aminosugars are included in this category of polar bases.

The disclosure includes products comprising salts of the formula (X)

where aa¹, aa², X and R¹ are as defined previously and A⁺ is theprotonated form of a pharmaceutically acceptable amine, as well as saltsin which both hydroxy groups of the boronate group are in salt form(preferably with another identical A⁺ group) and mixtures of such salts.In one class of such products, A⁺ is the protonated form of an aminedescribed in section 4B(i) below; in another class A⁺ is the protonatedform of an amine described in 4B(ii) below. Also included are productsin which R¹ is replaced by another R⁹ group.

Two illustrative classes of amine base are described in sections 4B(i)and 4B(ii) below. Particularly preferred are acid salts (in which one ofthe two boronic —OH groups is deprotonated). Most commonly, the saltscontain a single type of amine counter-ion (disregarding tracecontaminants) but the disclosure contemplates salts containing mixturesof amine counter-ions; in one sub-class, the different counter-ions allfall within the sub-section 4B(i) family described below or, as the casemay be, in the sub-section 4B(ii) family below; in another subclass, thesalts comprise a mixture of organic counter-ions which are not all fromthe same family (4B(i) or 1B(ii)).

4B(i) Aminosugars

-   -   The Identity of the aminosugar is not critical. Preferred        aminosugars include ring-opened sugars, especially glucamines.        Cyclic aminosugars are also envisaged as useful. One class of        the aminosugars is N-unsubstituted and another, preferred, class        is N-substituted by one or two N-substituents (e.g. one).        Suitable substituents are hydrocarbyl groups, for example and        without limitation containing from 1 to 12 carbon atoms; the        substituents may comprise alkyl or aryl moieties or both.        Exemplary substituents are C₁, C₂, C₃, C₄, C₅, C₆, C₇ and C₈        alkyl groups, In particular methyl and ethyl, of which methyl is        illustrative. Data indicate that aminosugars, especially        N-methyl-D-glucamine, are of surprisingly high solubility.    -   A most preferred aminosugar is N-methyl-D-glucamine:

4B(ii) Other Amines

-   -   Other suitable amines include amino acids (whether naturally        occurring or not) whose side chain is substituted by an amino        group, especially lysine.    -   Some amines are compounds of formula (XI):

-   -   where n, R² and R³ are as defined in relation to formula (IV).        The compounds of formula (VI) are usually of L-configuration.        The compounds of formula (VI) are lysine (n=4; R²=carboxyl;        R³═H) and lysine derivatives or analogues. A most preferred        amine is L-lysine.    -   Other suitable amines are nitrogen-containing heterocycles. At        least usually, such heterocyclic compounds are alicyclic; one        class of the heterocyclic compounds Is N-substituted and        another, preferred, class is N-unsubstituted. The heterocycles        may contain 6 ring-forming atoms, as in the cases of piperidine,        piperazine and morpholine. One class of amines includes        N-containing heterocycles substituted by polar substituents,        especially hydroxy, e.g. 1, 2 or 3 times.    -   The disclosure therefore includes amines other than aminosugars        which have one or more (e.g. 1, 2, 3, 4, 5 or 6) polar        substituents, especially hydroxy, in addition to one amine        group. Such compounds may have a ratio of (amino plus hydroxy        groups):carbon atoms of 1:2 to 1:1, the latter ratio being        particularly preferred.

The disclosure includes mixed salts, i.e. salts containing a mixture ofboropeptide moieties and/or counterions but single salts are preferred.

The salts in solid form may contain a solvent, e.g. water. There areIncluded a class of products in which the salts are essentiallyanhydrous. Also included is a class in which the salts are hydrates.

Use of the Products of the Disclosure

The salts of the disclosure are thrombin inhibitors. They are thereforeuseful for inhibiting thrombin. There are therefore provided compoundswhich have potential for controlling haemostasis and especially forinhibiting coagulation, for example in the treatment or prevention ofsecondary events after myocardial infarction. The medical use of thecompounds may be prophylactic (including to treat thrombosis as well asto prevent occurrence of thrombosis) as well as therapeutic (includingto prevent re-occurrence of thrombosis or secondary thrombotic events).

The salts may be employed when an anti-thrombogenic agent is needed.Further, it has been found that the salts, including those of boronicadds of Formula (II), are beneficial in that the class is useful fortreating arterial thrombosis by therapy or prophylaxis. The disclosedsalts are thus indicated in the treatment or prophylaxis of thrombosisand hypercoaguability in blood and tissues of animals including man. Theterm “thrombosis” includes inter alia atrophic thrombosis, arterialthrombosis, cardiac thrombosis, coronary thrombosis, creepingthrombosis, Infective thrombosis, mesenteric thrombosis, placentalthrombosis, propagating thrombosis, traumatic thrombosis and venousthrombosis.

It is known that hypercoaguability may lead to thromboembolic diseases.

Examples of venous thromboembolism which may be treated or preventedwith compounds of the disclosure include obstruction of a vein,obstruction of a lung artery (pulmonary embolism), deep vein thrombosis,thrombosis associated with cancer and cancer chemotherapy, thrombosisinherited with thrombophilic diseases such as Protein C deficiency,Protein S deficiency, antithrombin III deficiency, and Factor V Leiden,and thrombosis resulting from acquired thrombophilic disorders such assystemic lupus erythematosus (inflammatory connective tissue disease).Also with regard to venous thromboembolism, compounds of the disclosureare useful for maintaining patency of Indwelling catheters.

Examples of cardiogenic thromboembolism which may be treated orprevented with compounds of the disclosure include thromboembolic stroke(detached thrombus causing neurological affliction related to Impairedcerebral blood supply), cardiogenic thromboembolism associated withatrial fibrillation (rapid, irregular twitching of upper heart chambermuscular fibrils), cardiogenic thromboembolism associated withprosthetic heart valves such as mechanical heart valves, and cardiogenicthromboembolism associated with heart disease.

Examples of conditions involving arterial thrombosis include unstableangina (severe constrictive pain in chest of coronary origin),myocardial infarction (heart muscle cell death resulting frominsufficient blood supply), ischemic heart disease (local Ischemia dueto obstruction (such as by arterial narrowing) of blood supply),reocclusion during or after percutaneous transluminal coronaryangioplasty, restenosis after percutaneous transluminal coronaryangioplasty, occlusion of coronary artery bypass grafts, and occlusivecerebrovascular disease. Also with regard to arterio-venous (mixed)thrombosis, anti-thrombotic compounds of the disclosure are useful formaintaining patency in arteriovenous shunts.

Other conditions associated with hypercoaguability and thromboembolicdiseases which may be mentioned inherited or acquired deficiencies inheparin cofactor II, circulating antiphospholipid antibodies (Lupusanticoagulant), homocysteinemia, heparin induced thrombocytopenia anddefects in fibrinolysis.

Particular uses which may be mentioned include the therapeutic and/orprophylactic treatment of venous thrombosis and pulmonary embolism.Preferred indications envisaged for the products of the disclosure(notably the salts of TRI 50c) include:

-   -   Prevention of venous thromboembolic events (e.g. deep vein        thrombosis and/or pulmonary embolism). Examples include patients        undergoing orthopedic surgery such as total hip replacement,        total knee replacement, major hip or knee surgery; patients        undergoing general surgery at high risk for thrombosis, such as        abdominal or pelvic surgery for cancer; and in patients        bedridden for more than 3 days and with acute cardiac failure,        acute respiratory failure, infection.    -   Prevention of thrombosis in the haemodialysis circuit in        patients, in patients with end stage renal disease.    -   Prevention of cardiovascular events (death, myocardial        infarction, etc) in patients with end stage renal disease,        whether or not requiring haemodialysis sessions.    -   Prevention of venous thrombo-embolic events in patients        receiving chemotherapy through an indwelling catheter.    -   Prevention of thromboembolic events in patients undergoing lower        limb arterial reconstructive procedures (bypass,        endarteriectomy, transluminal angioplasty, etc).    -   Treatment of venous thromboembolic events.    -   Prevention of cardiovascular events in acute coronary syndromes        (e.g. unstable angina, non Q wave myocardial        ischaemia/infarction), in combination with another        cardiovascular agent, for example aspirin (acetylsalicylic acid;        aspirin is a registered trade mark in Germany), thrombolytics        (see below for examples), antiplatelet agents (see below for        examples).    -   Treatment of patients with acute myocardial infarction in        combination with acetylsalicylic acid, thrombolytics (see below        for examples).

The thrombin inhibitors of the disclosure are thus indicated both in thetherapeutic and/or prophylactic treatment of all the aforesaiddisorders.

In one method, the products of the disclosure are used for the treatmentof patients by haemodialysis, by providing the product in the dialysissolution, as described in relation to other thrombin inhibitors in WO00/41715. The disclosure therefore includes dialysing solutions anddialysing concentrates which comprise a product of the disclosure, aswell as a method of treatment by dialysis of a patient in need of suchtreatment, which method comprises the use of a dialysing solutionincluding a low molecular weight thrombin inhibitor. Also included isthe use of an anti-thrombotic product of the disclosure for themanufacture of a medicament for the treatment by dialysis of a patient,in which the anti-thrombotic product of the disclosure is provided inthe dialysing solution.

In another method, the products of the disclosure are used to combatundesirable cell proliferation, as described in relation to otherthrombin inhibitors in WO 01/41796. The undesirable cell proliferationis typically undesirable hyperplastic cell proliferation, for exampleproliferation of smooth muscle cells, especially vascular smooth musclecells. The products of the disclosure particularly find application inthe treatment of intimal hyperplasia, one component of which isproliferation of smooth muscle cells. Restenosis can be considered to bedue to neointimal hyperplasia; accordingly intimal hyperplasia in thecontext of the disclosure includes restenosis.

The products of the disclosure are also contemplated for the treatmentof Ischemic disorders. More particularly, they may be used in thetreatment (whether therapeutic or prophylactic) of an ischemic disorderin a patient having, or at risk of, non-valvular atrial fibrillation(NVAF) as described in relation to other thrombin inhibitors in WO02/36157. Ischemic disorders are conditions whose results include arestriction in blood flow to a part of the body. The term will beunderstood to include thrombosis and hypercoagulability in blood,tissues and/or organs. Particular uses that may be mentioned include theprevention and/or treatment of ischemic heart disease, myocardialinfarction, systemic embolic events in e.g. the kidneys or spleen, andmore particularly of cerebral ischemia, including cerebral thrombosis,cerebral embolism and/or cerebral ischemia associated with non-cerebralthrombosis or embolism (in other words the treatment (whethertherapeutic or prophylactic) of thrombotic or ischemic stroke and oftransient ischemic attack), particularly in patients with, or at riskof, NVAF.

The products of the disclosure are also contemplated for the treatmentof rheumatic/arthritic disorders, as described in relation to otherthrombin Inhibitors In WO 03/007984. Thus, the products of thedisclosure may be used in the treatment of chronic arthritis, rheumatoidarthritis, osteoarthritis or ankylosing spondylitis

Moreover, the products of the disclosure are expected to have utility inprophylaxis of re-occlusion (i.e. thrombosis) after thrombolysis,percutaneous trans-luminal angioplasty (PTA) and coronary bypassoperations; the prevention of re-thrombosis after microsurgery andvascular surgery in general. Further indications include the therapeuticand/or prophylactic treatment of disseminated intravascular coagulationcaused by bacteria, multiple trauma, intoxication or any othermechanism; anticoagulant treatment when blood is in contact with foreignsurfaces in the body such as vascular grafts, vascular stents, vascularcatheters, mechanical and biological prosthetic valves or any othermedical device; and anticoagulant treatment when blood is in contactwith medical devices outside the body such as during cardiovascularsurgery using a heart-lung machine or in haemodialysis.

The products of the disclosure are further indicated in the treatment ofconditions where there is an undesirable excess of thrombin withoutsigns of hypercoagulability, for example in neurodegenerative diseasessuch as Alzheimer's disease. In addition to its effects on thecoagulation process, thrombin is known to activate a large number ofcells (such as neutrophils, fibroblasts, endothelial cells and smoothmuscle cells). Therefore, the compounds of the disclosure may also beuseful for the therapeutic and/or prophylactic treatment of idiopathicand adult respiratory distress syndrome, pulmonary fibrosis followingtreatment with radiation or chemotherapy, septic shock, septicaemia,inflammatory responses, which include, but are not limited to, edema,acute or chronic atherosclerosis such as coronary arterial disease,cerebral arterial disease, peripheral arterial disease, reperfusiondamage, and restenosis after percutaneous trans-luminal angioplasty(PTA).

The salts may also be useful in the treatment of pancreatitis.

The salts described herein are further considered to be useful forinhibiting platelet procoagulant activity. The disclosure provides amethod for Inhibiting platelet pro-coagulant activity by administering asalt of a boronic acid described herein to a mammal at risk of, orsuffering from, arterial thrombosis, particularly a human patient. Alsoprovided is the use of such salts for the manufacture of medicaments forInhibiting platelet procoagulant activity.

The use of products of the disclosure as Inhibitors of plateletpro-coagulant activity is predicated on the observation that the boronicacids described herein are indicated to be effective at inhibitingarterial thrombosis as well as venous thrombosis.

Indications involving arterial thrombosis include acute coronarysyndromes (especially myocardial infarction and unstable angina),cerebrovascular thrombosis and peripheral arterial occlusion andarterial thrombosis occurring as a result of atrial fibrillation,valvular heart disease, arterio-venous shunts, indwelling catheters orcoronary stents. Accordingly, in another aspect there is provided amethod of treating a disease or condition selected from this group ofindications, comprising administering to a mammal, especially a humanpatient, a salt of the disclosure. The disclosure includes products foruse in an arterial environment, e.g. a coronary stent or other arterialimplant, having a coating which comprises a salt according to thedisclosure.

The salts of the disclosure may be used prophylactically to treat anindividual believed to be at risk of suffering from arterial thrombosisor a condition or disease involving arterial thrombosis ortherapeutically (including to prevent re-occurrence of thrombosis orsecondary thrombotic events).

There is therefore included the use of selective thrombin inhibitors(organoboronic add salts) described herein for treatment of the abovedisorders by prophylaxis or therapy as well as their use inpharmaceutical formulations and the manufacture of pharmaceuticalformulations.

Administration and Pharmaceutical Formulations

The salts may be administered to a host, for example, in the case wherethe drug has anti-thrombogenic activity, to obtain an anti-thrombogeniceffect in the case of larger animals, such as humans, the compounds maybe administered alone or in combination with pharmaceutically acceptablediluents, excipients or carriers. The term “pharmaceutically acceptable”includes acceptability for both human and veterinary purposes, of whichacceptability for human pharmaceutical use is preferred.

The salts of the disclosure may be combined and/or co-administered withany cardiovascular treatment agent. There are large numbers ofcardiovascular treatment agents available in commercial use, in clinicalevaluation and in pre-clinical development, which could be selected foruse with a product of the disclosure for the prevention ofcardiovascular disorders by combination drug therapy. Such agent can beone or more agents selected from, but not limited to several majorcategories, namely, a lipid-lowering drug, including an IBAT (ilealNa⁺/bile acid cotransporter) inhibitor, a fibrate, niacin, a statin, aCETP (cholesteryl ester transfer protein) inhibitor, and a bile addsequestrant, an anti-oxidant, including vitamin E and probucol, aIIb/IIIa antagonist (e.g. abciximab, eptifibatide, tirofiban), analdosterone Inhibitor (e.g. spirolactone and epoxymexrenone), anadenosine A2 receptor antagonist (e.g. losartan), an adenosine A3receptor agonist, a beta-blocker, acetylsalicylic add, a loop diureticand an ACE (angiotensin converting enzyme) inhibitor.

The salts of the disclosure may be combined and/or co-administered withany antithrombotic agent with a different mechanism of action, such asthe antiplatelet agents acetylsalicylic acid, ticlopidine, clopidogrel,thromboxane receptor and/or synthetase inhibitors, prostacyclin mimeticsand phosphodiesterase inhibitors and ADP-receptor (P₂ T) antagonists.

The products of the disclosure may further be combined and/orco-administered with thrombolytics such as tissue plasminogen activator(natural, recombinant or modified), streptokinase, urokinase,prourokinase, anisoylated plasminogen-streptokinase activator complex(APSAC), animal salivary gland plasminogen activators, and the like, inthe treatment of thrombotic diseases, in particular myocardialInfarction.

The salts of the disclosure may be combined and/or co-administered witha cardioprotectant, for example an adenosine A1 or A3 receptor agonist.

There is also provided a method for treating an inflammatory disease ina patient that comprises treating the patient with a product of thedisclosure and an NSAID, e.g., a COX-2 inhibitor. Such diseases includebut are not limited to nephritis, systemic lupus, erythematosus,rheumatoid arthritis, glomerulonephritis, vasculitis and sarcoidosis.Accordingly, the anti-thrombotic salts of the disclosure may be combinedand/or co-administered with an NSAID.

Typically, therefore, the salts described herein may be administered toa host to obtain a thrombin-inhibitory effect, or in any otherthrombin-inhibitory or anti-thrombotic context mentioned herein.

Actual dosage levels of active ingredients in the pharmaceuticalcompositions of this disclosure may be varied so as to obtain an amountof the active compound(s) that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration (referred to herein as a “therapeutically effectiveamount”). The selected dosage level will depend upon the activity of theparticular compound, the severity of the condition being treated and thecondition and prior medical history of the patient being treated.However, it is within the skill of the art to start doses of thecompound at levels lower than required for to achieve the desiredtherapeutic effect and to gradually increase the dosage until thedesired effect is achieved.

According to a further aspect there is provided a parenteral formulationincluding a salt as described herein. The formulation may consist of thesalt alone or it may contain additional components, in particular thesalt may be in combination with a pharmaceutically acceptable diluent,excipient or carrier, for example a tonicity agent for the purpose ofmaking the formulation substantially isotonic with the body of thesubject to receive the formulation, e.g. with human plasma. Theformulation may be in ready-to-use form or in a form requiringreconstitution prior to administration.

It is currently contemplated that, in the case of parenteraladministration, for example i.v. administration, of salts of TRI 50c,the salts might for instance be administered in an amount of from 0.5 to2.5 mg/Kg e.g. over a maximum period of 72 hours, calculated as TRI 50c.Other salts might be administered in equivalent molar amounts. Thedisclosure is not limited to administration in such quantities orregimens and includes dosages and regimens outside those described inthe previous sentence.

Parenteral preparations can be administered by one or more routes, suchas intravenous, subcutaneous, intradermal and infusion; a particularexample is intravenous. A formulation disclosed herein may beadministered using a syringe, injector, plunger for solid formulations,pump, or any other device recognized in the art for parenteraladministration.

Liquid dosage forms for parenteral administration may include solutions,suspensions, liposome formulations, or emulsions in oily or aqueousvehicles. In addition to the active compounds, the liquid dosage formsmay contain other compounds. Tonicity agents (for the purpose of makingthe formulations substantially isotonic with the subjects body, e.g.with human plasma) such as, for instance, sodium chloride, sodiumsulfate, dextrose, mannitol and/or glycerol may be optionally added tothe parenteral formulation. A pharmaceutically acceptable buffer may beadded to control pH. Thickening or viscosity agents, for instance wellknown cellulose derivatives (e.g. methylcellulose,carboxymethylcellulose, hydroxyethylcellulose andhydroxypropylmethylcellulose), gelatin and/or acacia, may optionally beadded to the parenteral formulation.

Solid dosage forms for parenteral administration may encompass solid andsemi-solid forms and may include pellets, powders, granules, patches,and gels. In such solid dosage forms, the active compound Is typicallymixed with at least one Inert, pharmaceutically acceptable excipient orcarrier. The disclosed salts may be presented as solids in finelydivided solid form, for example they may be milled or micronised.

The formulations may also include antioxidants and/or preservatives. Asantioxidants may be mentioned thiol derivatives (e.g, thioglycerol,cysteine, acetylcysteine, cystine, dithioerythreitol, dithiothreitol,gluthathione), tocopherols, butylated hydroxyanisole, butylatedhydroxytoluene, sulfurous acid salts (e.g. sodium sulfate, sodiumbisulfite, acetone sodium bisulfite, sodium metabisulfite, sodiumsulfite, sodium formaldehyde sulfoxylate, sodium thiosulfate) andnordihydroguaiareticacid. Suitable preservatives may for instance bephenol, chlorobutanol, benzylalcohol, methyl paraben, propyl paraben,benzalkonium chloride and cetylpyridinium chloride.

The parenteral formulations may be prepared as large volume parenterals(LVPs), e.g. larger than 100 ml, more particularly about 250 ml, of aliquid formulation of the active compound. Examples of LVPs are infusionbags. The parenteral formulations may alternatively be prepared as smallvolume parenterals (SVPs), e.g. about 100 ml or less of a liquidformulation of the active compound. Examples of SVPs are vials withsolution, vials for reconstitution, prefilled syringes for injection anddual chamber syringe devices.

The formulations of the disclosure include those in which the salt is analkali metal salt, for example a lithium, sodium or potassium salt, ofwhich sodium salts may be mentioned as particular salts. Another classof formulations contains aminosugar salts of the disclosed boronic adds,for example N-methyl-D-glucamine salts. The salts mentioned in thisparagraph may be administered as solutions in water, typicallycontaining one or more additives, for example isotonicity agent(s)and/or antioxidant(s). A suitable way to store the salts is in solidform, for example as dry powder, and to make them up into solutions foradministration prior to administration.

One class of formulations disclosed herein is intravenous formulations.For intravenously administered formulations, the active compound orcompounds can be present at varying concentrations, with a carrieracceptable for parenteral preparations making up the remainder.Particularly, the carrier is water, particularly pyrogen free water, oris aqueous based. Particularly, the carrier for such parenteralpreparations is an aqueous solution comprising a tonicity agent, forexample a sodium chloride solution.

By “aqueous based” is meant that formulation comprises a solvent whichconsists of water or of water and water-miscible organic solvent orsolvents; as well as containing a salt of disclosure in dissolved form,the solvent may have dissolved therein one or more other substances, forexample an antioxidant and/or an isotonicity agent. As organiccosolvents may be mentioned those water-miscible solvents commonly usedin the art, for example propyleneglycol, polyethyleneglycol 300,polyethyleneglycol 400 and ethanol. Preferably, organic co-solvents areonly used in cases where the active agent is not sufficiently soluble inwater for a therapeutically effective amount to be provided in a singledosage form. As previously indicated, the disclosure includesformulations of alkali metal salts of the disclosed boronic acids, e.g.TRI 50c, having a solvent which consists of water.

The solubility of the active compound in the present formulations may besuch that the turbidity of the formulation is lower than 50 NTU, e.g.lower than 20 NTU such as lower than 10 NTU.

It is desirable that parenteral formulations are administered at or nearphysiological pH. It is believed that administration in a formulation ata high pH (i.e., greater than 8) or at a low pH (i.e., less than 5) isundesirable. In particular, it is contemplated that the formulationswould be administered at a pH of between 6.0 and 7.0 such as a pH of6.5.

The parenteral formulation may be purged of air when being packaged. Theparenteral formulation may be packaged in a sterile container, e.g.vial, as a solution, suspension, gel, emulsion, solid or a powder. Suchformulations may be stored either in ready-to-use form or in a formrequiring reconstitution prior to administration.

Parenteral formulations according to the disclosure may be packaged incontainers. Containers may be chosen which are made of material which isnon-reactive or substantially non-reactive with the parenteralformulation. Glass containers or plastics containers, e.g. plasticsinfusion bags, may be used. A concern of container systems is theprotection they afford a solution against UV degradation. If desired,amber glass employing iron oxide or an opaque cover fitted over thecontainer may afford the appropriate UV protection.

Plastics containers such as plastics infusion bags are advantageous inthat they are relatively light weight and non-breakable and thus moreeasily stored. This is particularly the case for Large Volumeparenterals.

The intravenous preparations may be prepared by combining the activecompound or compounds with the carrier. After the formulation is mixed,it may be sterilized, for example using known methods. Once theformulation has been sterilized, it is ready to be administered orpackaged, particularly in dark packaging (e.g. bottles or plasticspackaging), for storage. It is envisaged, however, that the disclosedsalts might not be stored in solution but as dry solids, particularly afinely divided form such as, for example, a lyophilisate, in order toprolong shelf life; this would of course apply to other parenteralformulations, not only intravenous ones.

The intravenous preparations may take the form of large volumeparenterals or of small volume parenterals, as described above.

In a specific embodiment, the present disclosure is directed toproducts, particularly kits, for producing a single-dose administrationunit. The products (kits) may each contain both a first container havingthe active compound (optionally combined with additives, for exampleanti-oxidant, preservative and, in some Instances, tonicity agent) and asecond container having the carrier/diluent (for example water,optionally containing one or more additives, for example tonicityagent). As examples of such products may be mentioned single andmulti-chambered (e.g. dual-chamber) pre-filled syringes; exemplarypre-filled syringes are available from Vetter GmbH, Ravensburg, Germany.Such dual chamber syringes or binary syringes will have in one chamber adry preparation including or consisting of the active compound and inanother chamber a suitable carrier or diluent such as described herein.The two chambers are joined in such a way that the solid and the liquidmix to form the final solution.

One class of formulations disclosed herein comprises subcutaneous orintradermal formulations (for example formulations for injection) inwhich the active salt (or active agent combination) is formulated into aparenteral preparation that can be injected subcutaneously orintradermally. The formulation for administration will comprise theactive salt and a liquid carrier.

The carrier utilized in a parenteral preparation that will be injectedsubcutaneously or intradermally may be an aqueous carrier (for examplewater, typically containing an additive e.g. an antioxidant and/or anisotonicity agent) or a nonaqueous carrier (again one or more additivesmay be incorporated). As a non-aqueous carrier for such parenteralpreparations may be mentioned highly purified olive oil.

The active compound and the carrier are typically combined, for examplein a mixer. After the formulation is mixed, it is preferably sterilized,such as with U.V. radiation. Once the formulation has been sterilized,it is ready to be injected or packaged for storage. It is envisaged,however, that the disclosed salts will not be stored in liquidformulation but as dry solids, in order to prolong shelf life.

For making subcutaneous implants, the active salt may suitably beformulated together with one or more polymers that are gradually erodedor degraded when in use, e.g. silicone polymers, ethylene vinylacetate,polyethylene or polypropylene.

Transdermal formulations may be prepared in the form of matrices ormembranes, or as fluid or viscous formulations in oil or hydrogels or asa compressed powder pellet. For transdermal patches, an adhesive whichis compatible with the skin may be included, such as polyacrylate, asilicone adhesive or polyisobutylene, as well as a foil made of, e.g.,polyethylene, polypropylene, ethylene vinylacetate, polyvinylchloride,polyvinylidene chloride or polyester, and a removable protective foilmade from, e.g., polyester or paper coated with silicone or afluoropolymer. For the preparation of transdermal solutions or gels,water or organic solvents or mixtures thereof may be used. Transdermalgels may furthermore contain one or more suitable gelling agents orthickeners such as silicone, tragacanth, starch or starch derivatives,cellulose or cellulose derivatives or polyacrylic acids or derivativesthereof. Transdermal formulations may also suitably contain one or moresubstances that enhance absorption though the skin, such as bile saltsor derivatives thereof and/or phospholipids. Transdermal formulationsmay be prepared according to a method disclosed in, e.g., B W Barry,“Dermatological Formulations, Percutaneous Absorption”, Marcel DekkerInc., New York—Basel, 1983, or Y W Chien, “Transdermal ControlledSystemic Medications”, Marcel Dekker Inc., New York—Basel, 1987.

It will be understood from the aforegoing that there are providedpharmaceutical products comprising an alkali metal salt, particularlysodium salt, of a boronic acid of Formula (I) in dry fine particle form,suitable for reconstitution into an aqueous read-to-use parenteralformulation. The alkali metal salt is suitably an acid salt. The alkalimetal salt may be in a small volume parenteral unit dosage form. Thealkali metal salt may be presented in a form, e.g. dry powder form,suitable for reconstituting as a large volume parenteral. One example isa sodium salt of a boronic acid of Formula (I), particularly TRI 50c, indry powder form for reconstitution as a liquid intravenous formulation(solution) containing a tonicity agent, particularly sodium chloride.The dry powder form of a salt used in a parenteral formulation may be alyophilisate. The reconstituted solution may be administered byinjection or infusion.

Synthesis 1. Peptide/Peptidomimetic Synthesis

The synthesis of boropeptides, including, for example,Cbz-D-Phe-Pro-BoroMpg-OPinacol is familiar to those skilled in the artand described in the prior art mentioned above, including Claeson et al.(U.S. Pat. No. 5,574,014 and others) and Kakkar et al. (WO 92/07869 andfamily members including U.S. Pat. No. 5,648,338). It is described alsoby Elgendy et al Adv. Exp. Med. Biol. (USA) 340:173-178, 1993; Claeson,G. et al Biochem.J. 290:309-312, 1993; Deadman et al J. EnzymeInhibition 9:29-41, 1995, and by Deadman et al J. Med. Chem.38:1511-1522, 1995.

Stereoselective synthesis with S or R configuration at the chiralB-terminal carbon may be conducted using established methodology(Elgendy et al Tetrahedron. Lett. 33:4209-4212, 1992; WO 92/07869 andfamily members including U.S. Pat. No. 5,648,338) using (+) or(−)-pinanediol as the chiral director (Matteson et al J. Am. Chem. Soc.108:810-819, 1986; Matteson et al Organometallics. 3:1284-1288, 1984).Another approach is to resolve the requisite aminoboronate intermediate(e.g. Mpg-BOPinacol) to selectively obtain the desired (R)-isomer andcouple it to the dipeptide moiety (e.g. Cbz-(R)-Phe-(S)—Pro, which isthe same as Cbz-D-Phe-L-Pro) which will form the remainder of themolecule.

The boropeptides may be synthesised Initially in the form of boronicacid esters, particularly esters with diols. Such diol esters may beconverted to the peptide boronic acid as described next.

2. Ester to Acid Conversion

A peptide boronate ester such as Cbz-(R)-Phe-Pro-BoroMpg-OPinacol may behydrolysed to form the corresponding acid.

A novel technique for converting a diol ester of a peptide boronic addof formula (I) into the acid comprises dissolving the diol ester in anether and particularly a dialkyl ether, reacting the thus-dissolved diolwith a diolamine, for example a dialkanolamine, to form a productprecipitate, recovering the precipitate, dissolving it in a polarorganic solvent and reacting the thus-dissolved product with an aqueousmedium, e.g. an aqueous add, to form the peptide boronic acid. Theboronic add may be recovered from the organic layer of the mixtureresulting from the reaction, for example by removing the solvent, e.g.by evaporation under vacuum or distillation. The reaction between thediol ester and the diolamine may be carried out under reflux, forexample.

The identity of the diol is not critical. As suitable diols may bementioned aliphatic and aromatic compounds having hydroxy groups thatare substituted on adjacent carbon atoms or on carbon atoms substitutedby another carbon. That is to say, suitable diols include compoundshaving at least two hydroxy groups separated by at least two connectingcarbon atoms in a chain or ring. One class of diols compriseshydrocarbons substituted by exactly two hydroxy groups. One such diol ispinacol and another is pinanediol; there may also be mentionedneopentylglycol, 1,2-ethanediol, 1,2-propanedlol, 1,3-propanediol,2,3-butanediol, 1,2-diisopropylethanediol, 5,6-decanediol and1,2-dicyclohexylethanediol.

The alkyl groups of the dialkyl ether preferably have 1, 2, 3 or 4carbon atoms and the alkyl groups may be the same or different. Anexemplary ether is diethyl ether.

The alkyl groups of the dialkanolamine preferably have 1, 2, 3 or 4carbon atoms and the alkyl groups may be the same or different. Anexemplary dialkanolamine is diethanolamine. The diethanolamine/boronicacid reaction product hydrolyses in water at room temperature and therate of hydrolysis may be accelerated by adding acid or base.

The polar organic solvent is preferably CHCl₃. Other examples arepolyhalogenated alkanes generally and ethyl acetate. In principle, anypolar organic solvent is acceptable other than alcohols.

The aqueous add is suitably a strong inorganic acid at a pH in theregion of 1 such as hydrochloric acid, for example.

After reaction with the acid, the reaction mixture is suitably washedwith, for example, NH₄Cl or another mild base.

An Example of a Specific Procedure is as Follows

1. The pinacol or pinanediol ester of the selected peptide boronic acidis dissolved in diethylether.2. Diethanolamine is added and the mixture is refluxed at 40° C.3. The precipitated product is removed (filtered), washed (usuallyseveral times) with diethyl ether or another polar organic solvent otherthan an alcohol, and dried (e.g. by evaporation under vacuum).4. The dry product is dissolved in a polar organic solvent other than analcohol, e.g. CHCl₃. Aqueous acid or base is added, e.g. hydrochloricacid (pH 1), and the mixture is stirred for e.g. approximately 1 h atroom temperature.5. The organic layer is removed and washed with NH₄Cl solution.6. The organic solvent is distilled off and the residual solid productis dried.

The above process results in the formation of what may conveniently bereferred to as a “diolamine adduct” of the peptide boronic adds offormula (I), especially such adducts with diethanolamine, and suchadducts are themselves included in the disclosure. The molecularstructure of such adducts is not known: they might comprise a compoundin which the two oxygens and the nitrogen of the diolamine are allcoordinated to the boron; they might comprise ions. The adducts arehowever considered to be esters. A particular novel product included inthe disclosure is that obtainable by reacting a pinacol or pinanediolester of a compound of Formula VIII, particularly (R,S,R)-TRI 50c, anddiethanolamine, i.e. the novel product is an (R,S,R)-TRI50c/diethanolamine “adduct” where the acid is (R,S,R)-TRI 50c.

The diolamine materials of the disclosure may be defined as acomposition of matter comprising:

-   -   (i) a species of formula (XII)

wherein X is H or an amino protecting group, the boron atom isoptionally coordinated additionally with a nitrogen atom, and thevalency status of the terminal oxygens is open (they may be attached toa second covalent bond, be ionised as —O⁻, or have some other, forexample intermediate, status); and, In bonding association therewith

-   -   (ii) a species of formula (XIII)

wherein the valency status of the nitrogen atom and the two oxygen atomsis open. It will be appreciated that the terminal oxygen atoms of thespecies of formula (IX) and the oxygen atoms of the species of formula(X) may be the same oxygen atoms, in which case the species of formula(X) forms a diol ester with the species of formula (IX).

It will be appreciated that the aforegoing technique comprises anexample of a method for recovering an organoboronic acid product, themethod comprising providing in a solvent a dissolved mixture comprisingthe organoboronic acid in a soluble form and a compound having twohydroxy groups and an amino group (i.e. a diolamine), causing orallowing the organoboronic acid and the diolamine to react to form aprecipitate, and recovering the precipitate. The soluble form of theorganoboronic acid may be a diol ester, as discussed above. The solventmay be an ether, as discussed above. The organoboronic acid may be oneof the organoboronic adds referred to in this specification, for exampleit may be of Formula (I) or (III). The method described in thisparagraph is novel and forms an aspect of the disclosure. A recoverymethod is filtration.

The reaction between the diolamine and the soluble form of theorganoboronic acid is suitable carried out at an elevated temperature,for example under reflux.

Another aspect of the disclosure is a method for recovering anorganoboron species, comprising

-   -   providing, in a form soluble in an ether, an organoboronic acid,        for example a drug such as, e.g., a compound of formula (III);    -   forming a solution of the soluble form in the ether;    -   combining the solution with a dialkanolamine and allowing or        causing the dialkanolamine to react with the soluble form of the        organoboronic acid to form an insoluble precipitate; and    -   recovering the precipitate.

The term “soluble” in the preceding paragraph refers to species whichare substantially more soluble In the reaction medium than is theprecipitated product. In variants of the method, the ether is replacedby toluene or another aromatic solvent.

The diethanolamine precipitation technique described above is an exampleof another novel method, which is a method for recovering from ethersolution a pinacol or pinanediol ester of a peptide boronic acid,comprising dissolving diethanolamine in the solution, allowing orcausing a precipitate to form and recovering the precipitate. Thedisclosure encompasses variants of this methods in which another diolthan pinacol or pinanediol is used.

The precipitated material, i.e. the “adduct”, may be converted into thefree organoboronic acid, for example by contacting it with an acid. Theacid may be an aqueous acid, for example an aqueous Inorganic acid, e.g.as described above. The precipitate may be dissolved, for example 1n anorganic solvent, prior to being contacted with the acid.

The disclosure therefore provides a method for making an organoboronicacid, comprising converting its diolamine reaction product to the add.

The acid resulting from the methods described in the previous twoparagraphs may be converted to a salt of the acid with a multivalentmetal, which salt may in turn be formulated into a pharmaceuticalcomposition in parenteral dosage form.

3. Salt Synthesis

In general, the salts may be prepared by contacting the relevant peptideboronic acid with a strong base appropriate to form the desired salt. Inthe case of metal salts, the metal hydroxides are suitable bases(alternatively, metal carbonates might be used, for example), whilstsometimes it is more convenient to contact the acid with a relevantmetal alkoxide (eg methoxide), for which purpose the correspondingalkanol is a suitable solvent. Salts with organic bases may be preparedby contacting the peptide boronic acid with the organic base itself.Illustrative salts are acid salts (one —BOH proton replaced) and, tomake add salts with a monovalent cation, the acid and the base aresuitably reacted in substantially equimolar quantities. Generallystated, therefore, the usual acid:base molar ratio is substantially n:1,where n is the valency of the cation of the base.

In one procedure, a solution of the peptide boronic acid in awater-miscible organic solvent, for example acetonitrile or an alcohol(e.g. ethanol, methanol, a propanol, for example iso-propanol, oranother alkanol), is combined with an aqueous solution of the base. Theacid and the base are allowed to react and the salt is recovered. Thereaction is typically carried out at ambient temperature (e.g. at atemperature of from 15 to 30° C., e.g. 15 to 25° C.), but an elevatedtemperature may be used, for example up to the boiling point of thereaction mixture but more usually lower, e.g. a temperature of up to 40°C. or 50° C. The reaction mixture may be allowed to stand or be agitated(usually stirred).

The time during which the acid and the base are allowed to react is notcritical but it has been found desirable to maintain the reactionmixture for at least one hour. A period of from one to two hours isusually suitable but longer reaction times may be employed.

The salt may be recovered from the reaction mixture by any suitablemethod, for example evaporation or precipitation. Precipitation may becarried out by adding an excess of a miscible solvent in which the salthas limited solubility. In one preferred technique, the salt isrecovered by evacuating the reaction mixture to dryness. The salt ispreferably thereafter purified, for example by redissolving the saltbefore filtering the resulting solution and drying it, for example byevacuating it to dryness. The redissolution may be performed usingwater, e.g. distilled water. The salt may then be further purified, forexample in order to remove residual water by further redissolution in asuitable solvent, which is advantageously ethyl acetate or THF followedby evaporating to dryness. The purification procedure may be carried outat ambient temperature (say, 15 to 30° C., e.g. 15 to 25° C.), or at amodestly elevated temperature, such as e.g. a temperature not exceeding40° C. or 50° C.; for example the salt may be dissolved in water and/orsolvent by agitating with or without warming to, for example, 37° C.

Also included is a method for drying the salts of the disclosure andother peptide boronic acid salts, comprising dissolving them in anorganic solvent, e.g. ethyl acetate or THF, and then evaporating todryness, e.g. by evacuation.

Generally, preferred solvents for use in purifying the salts are ethylacetate or THF, or perhaps another organic solvent.

A general procedure for synthesising salts of Cbz-Phe-Pro-BoroMpg-OH isas follows:

Cbz-Phe-Pro-BoroMpg-OH (20.00 g, 38.1 mM) is dissolved in acetonitrile(200 ml) with stirring at room temperature. To this solution is addedthe requisite base in solution in distilled water (190 ml); the base isadded as a 0.2M solution for a monovalent cation. The resultant clearsolution is allowed to react for example by being left to stand or beingagitated, for a usual period, in either case, of from one to two hours.The reaction is typically carried out at ambient temperature (e.g.15-30° C., e.g. 15 to 25° C.) but alternatively the temperature may beelevated (e.g. up to 30° C., 40° C. or 50° C.). The reaction mixture isthen evacuated to dryness under vacuum with its temperature notexceeding 37° C., typically to yield a white brittle solid or anoil/tacky liquid. The oil/tacky liquid is redissolved in the minimumamount of distilled water necessary (200 ml to 4 L), typically withwarming (e.g. to 30-40° C.), usually for up to 2 hours. The solution isfiltered, suitably through filter paper, and evacuated to dryness, againwith the temperature of the solution not exceeding 37° C., or freezedried. The resultant product is dried under vacuum overnight to normallyyield a white brittle solid. If the product is present as an oil ortacky solid then it is dissolved in ethyl acetate and evacuated todryness to produce the product as a white solid. The white solid istypically a coarse, amorphous powder.

In variations of the aforegoing general procedure, the acetonitrile isreplaced by another water-miscible organic solvent, notably an alcohol,as discussed above, especially ethanol, methanol, iso-propanol oranother propanol.

Where a boronic acid salt is less soluble in a selected reaction mediumfor salt formation such that its direct preparation from thecorresponding acid and base is inconvenient, the less soluble salt maybe prepared from a salt more soluble in the reaction medium.

There is provided also the use of a boronic acid to make a salt of thedisclosure. Included also is a method of preparing a product of thedisclosure, comprising contacting a boronic acid, e.g. of formula (I),(II) or (III), with a base capable of making such a salt.

The peptide boronic acid of formula (I) used to prepare thepharmaceutical preparations is typically of GLP or GMP quality, or incompliance with GLP (good laboratory practice) or GMP (goodmanufacturing practice); such acids are included in the disclosure.

Similarly the acids are usually sterile and/or acceptable forpharmaceutical use, and one aspect of the disclosure reside in acomposition of matter which is sterile or acceptable for pharmaceuticaluse, or both, and comprises a peptide boronic acid of formula (I). Sucha composition of matter may be in particulate form or in the form of aliquid solution or dispersion.

The intermediate acid may be in isolated form and such isolated acidsare included in the disclosure, especially Isolated acids which are apeptide boronic acid of formula (VIII):

X—(R)-Phe-(S)—Pro-(R)-Mpg-B(OH)₂  (VIII)

wherein X is H (to form NH₂) or an amino-protecting group.

One typical way of providing the intermediate acids is as a particulatecomposition consisting predominantly of such a peptide boronic acid, andthese compositions are included in the disclosure. The peptide boronicadd often forms at least 75% by weight of the composition and typicallyat least 85% by weight of the composition, e.g. at least 95% by weightof the composition.

Another typical way of providing the intermediate acids is as a liquidcomposition consisting of, or consisting essentially of, a peptideboronic acid of formula (II) and a liquid vehicle in which it isdissolved or suspended. The liquid vehicle may be an aqueous medium,e.g. water, or an alcohol, for example methanol, ethanol, isopropanol,or another propanol, another alkanol or a mixture of the aforegoing.

The compositions of the Intermediate acids are generally sterile. Thecompositions may contain the peptide boronic acid in finely dividedform, to facilitate further processing.

Separation of Stereoisomers

The stereoisomers of a peptide boronic ester or a synthetic intermediateaminoboronate may be resolved in, for example, any known way. Inparticular, stereoisomers of boronic esters may be resolved by HPLC.

EXAMPLES Examples 1 to 4 Introductory Remarks Apparatus

Throughout the following procedures of Examples 1 to 4, standardlaboratory glassware and, where appropriate, specialised apparatus forhandling and transferring of air sensitive reagents are used.

All glassware is heated at 140-160° C. for at least 4 hours before useand then cooled either in a desiccator or by assembling hot and purgingwith a stream of dry nitrogen.

Solvents

The organic solvents used in the procedures of Examples 1 to 4 are alldry. Suitably, they are dried over sodium wire before use.

Dryness

In the drying procedures of Example 1 to 4, products are tested fordryness (including dryness in terms of organic solvent) by observingweight loss on drying. The following procedure was followed to determineloss on drying: a sample was placed in a vacuum drier and dried at 40°C. at 100 mbar for 2 hours. Products are considered dry when thedecrease in weight upon drying is less than 0.5% of the total weight ofthe starting material.

Examples 1 to 4 describe performance of the following reaction schemeand conversion of the resultant TRI 50c to sodium and calcium saltsthereof:

Example 1 Synthesis Of TRI 50D Step 1: Z-DIPIN B Procedure A

17.8 g (732.5 mmole) magnesium turnings, 0.1 g (0.4 mmole) iodine and127 ml dry tetrahydrofuran are charged and heated to reflux. Then 15 mlof a solution of 66 g (608 mmole) 1-chloro-3-methoxypropane in 185 mldry tetrahydrofuran are added and stirred under reflux until thevigorous reaction starts. After the initial exotherm ceases, thesolution of 1-chloro-3-methoxypropane is added slowly to maintain gentlereflux until all the magnesium is consumed. After the reaction isfinished, the reaction mixture is cooled to ambient temperature andslowly added to a solution of 64.4 g (620 mmole) trimethylborate in 95ml dry tetrahydrofuran; the latter solution is cooled to below 0° C.and, if it warms up during the course of the reaction, the reactionmixture must be added to it sufficiently slowly to maintain thetemperature of this solution below 65° C. Upon complete addition, thereaction mixture is allowed to warm to about 0° C. and stirred foranother 60 minutes. Then a solution of 22.4 ml sulfuric acid in 400 mlwater is added slowly so as to maintain the temperature below 20° C. Thelayers are allowed to settle and the phases are separated. The aqueouslayer is rewashed three times with 200 ml tert.-butylmethylether. Thecombined organic layers are allowed to settle and additional waterseparated from this solution is removed. The organic layer is dried overmagnesium sulfate, filtered and evaporated to dryness. The evaporationresidue is filtered from the precipitated solid and the filtratedissolved in 175 ml toluene. 34.8 g (292 mmole) pinacol is charged tothe solution followed by stirring at ambient temperature for not lessthan 10 hours. The solution is evaporated to dryness, dissolved in 475ml n-heptane and washed three times with 290 ml saturated aqueoussolution of sodium hydrogen carbonate. The n-heptane solution isevaporated to dryness and the evaporation residue distilled and thefraction with Bp 40-50° C. at 0.1-0.5 mbar recovered.

Boiling point: 40-50° C./0.1-0.5 mbar

Yield: 40.9 g (70%) Z-DIPIN B (oil)

Procedure B

17.8 g (732.5 mmole) magnesium turnings, 0.1 g (0.4 mmole) iodine and127 ml dry tetrahydrofuran are charged and heated to reflux. Then 15 mlof a solution of 66 g (608 mmole) 1-chloro-3-methoxypropane in 185 mldry tetrahydrofuran are added and stirred under reflux until thevigorous reaction starts. After the Initial exotherm ceases, thesolution of 1-chloro-3-methoxypropane is added slowly to maintain gentlereflux. After the reaction is finished, the reaction mixture is cooledto ambient temperature and slowly added to a solution of 64.4 g (620mmole) trimethylborate in 95 ml dry tetrahydrofuran, maintaining thetemperature of this solution below minus 65° C. Upon complete addition,the reaction mixture is allowed to warm to about 0° C. and stirred foranother 60 minutes. Then a solution of 22.4 ml sulfuric acid in 400 mlwater is added slowly so as to maintain the temperature below 20° C. Theorganic solvent is removed by distillation under vacuum. 300 mln-heptane is charged to the aqueous solution of the evaporation residuefollowed by addition of 34.8 g (292 mmole) pinacol. Thetwo-phase-mixture is stirred at ambient temperature for not less than 2hours. After allowing the layers to settle, the aqueous phase isseparated. 300 ml n-heptane is charged to the aqueous solution and thetwo-phase-mixture is stirred at ambient temperature for not less than 2hours. After allowing the layers to settle, the aqueous phase isseparated. The organic layers are combined and washed once with 200 mlwater, followed by 200 ml saturated sodium hydrogen carbonate solutionand two further washes with 200 ml water each. The n-heptane solution isevaporated to dryness and the evaporation residue distilled and thefraction with Bp 40-50° C. at 0.1-0.5 mbar recovered.

Boiling point: 40-50° C./0.1-0.5 mbar

Yield: 40.9 g (70-85%) Z-DIPIN 8 (oil)

Step 2: Z-DIPIN C

16.6 g (164 mmole) diisopropylamine and 220 ml tetrahydrofuran arecharged and cooled to −30 to −40° C. To this solution 41.8 g (163 mmole)n-butyl lithium, 25% in n-heptane is added, followed by stirring at 0 to−5% for one hour. This freshly prepared solution of lithiumdiisopropylamide is cooled to −30° C. and then added to a solution of27.9 g (139 mmole) Z-DIPIN B in 120 ml tetrahydrofuran and 35.5 g (418mmole) dichloromethane at a temperature between −60 and −75° C. Thesolution is stirred at that temperature for half an hour followed byaddition of 480 ml (240 mmole) 0.5N anhydrous Zinc(II)-chloride intetrahydrofuran or 32.5 g (240 mmole) anhydrous solid Zinc(II)-chloride.After stirring at −65° C. for one hour, the reaction mixture is allowedto warm to ambient temperature and stirred for another 16-18 hours. Thereaction mixture is evaporated to dryness (i.e. until solvent isremoved) and followed by addition of 385 ml r-heptane. The reactionmixture is washed with 150 ml 5% sulfuric acid, with 190 ml saturatedsodium hydrogen carbonate solution, and 180 ml saturated sodium chloridesolution. The organic layer is dried over magnesium sulfate, filteredand evaporated to dryness (i.e. until solvent is removed). The oilyresidue is transferred into the next step without further purification.

Yield: 19 g (55%) Z-DIPIN C

Step 3: Z-DIPIN D

To a solution of 23.8 g (148 mmole) hexamethyldisilazane in 400 mltetrahydrofuran at −15° C. is added 34.7 g (136 mmole) n-butyl lithium,25% in n-heptane and stirred for one hour. The solution is cooled to−55° C. followed by the addition of 30.6 g (123 mmole) Z-DIPIN Cdissolved in 290 ml tetrahydrofuran and 35 ml tetrahydrofuran to thisfreshly prepared solution of LiHMDS. The solution is allowed to warm toambient temperature and stirred for 12 hours. The reaction mixture isevaporated to dryness, the evaporation residue dissolved in 174 mln-heptane, washed with 170 ml water and 75 ml saturated sodium chloridesolution. The organic phase is dried over magnesium sulfate, filteredand evaporated to complete dryness (i.e. until solvent is removed). Theoily residue is dissolved in 100 g n-heptane. This solution is carriedover into the next step without further purification.

Yield: 32.2 g (700%) Z-DIPIN D

Step 4: Z-DIPIN (TRI 50b, crude)

A solution of 26.6 g (71 mmole) Z-DIPIN D in 82.6 g n-heptane is dilutedwith 60 ml n-heptane and cooled to −60° C. followed by introduction of10.5 g (285 mmole) hydrogen chloride. The reaction mixture issubsequently evacuated and flushed with nitrogen, while the temperatureis increased in increments of about 20° C. to ambient temperature. Thesolvent is removed from the oily precipitate and replaced several timesby 60 ml fresh n-heptane. The oily residue is dissolved in 60 mltetrahydrofuran (Solution A).

To a different flask 130 ml tetrahydrofuran, 24.5 g (61.5 mmole)Z-D-Phe-Pro-OH and 6.22 g (61.5 mmole) N-methylmorpholine are chargedand cooled to −20° C. To this solution a solution of 8.4 g (61.5 mmole)isobutylchloroformate in 20 ml tetrahydrofuran is added and stirred for30 minutes, followed by addition of Solution A at −25° C. Upon completeaddition, up to 16 ml (115 mmole) triethylamine is added to adjust thepH to 9-10, measured using a pH stick. The reaction mixture is allowedto warm to ambient temperature and stirred for 3 hours, still undernitrogen. The solvent is evaporated to dryness and the evaporationresidue dissolved in 340 ml tert.-butylmethylether (t-BME). The solutionof Z-DIPIN in t-BME is washed twice with 175 ml 1.5% hydrochloric acid.The combined acidic washes are given a rewash with 175 ml t-BME. Thecombined organic layers are washed with 175 ml water, with 175 mlsaturated sodium hydrogen carbonate solution, with 175 ml 25% sodiumchloride solution, dried over magnesium sulfate and filtered. Thissolution is carried over into the next step without furtherpurification.

Yield: 29.9 g (80%) Z-DIPIN

Example 2 Synthesis of TRI 50D (Diethanolamine Adduct of Tri 50C)

The starting material used in this Example is the solution of TRI 50b(“Z-DIPIN”) obtained in Example 1. The solution is carried forward tothe synthesis of TRI 50d without further purification. The solution ofZ-DIPIN in t-BME (containing 7.0 g (11.5 mmole) (R,S,R) TRI 50b,calculated based on HPLC results of Z-DIPIN) is evaporated to drynessand the evaporation residue dissolved in 80 ml diethylether. 1.51 g(14.4 mmole) diethanolamine is added and the mixture heated at refluxfor at least 10 hours, during which process the product precipitates.The suspension is cooled to 5-10° C., filtered and the filter residuewashed with diethylether.

To improve chiral and chemical purity the wet filter cake (7 g) isdissolved in 7 ml dichloromethane, cooled to 0-5° C. and the productprecipitated by addition of 42 ml diethylether and filtered. Theisolated wet product is dried at 35° C. in vacuum or at least 4 hours,until day.

Yield: 5.5 g (80%) Tri50d

Melting Point: 140-145° C.

Example 3 Preparation of Sodium Salt of TRI 50C

1.5 kg (2.5 mole) TRI 50d from Example 2 is dissolved in 10.5 Ldichloromethane. 11 L 2% hydrochloric acid is added and the mixture isstirred for at most 30 minutes (optimally about 20 minutes) at roomtemperature. A predpitate forms in the organic phase. After stirring,the layers are allowed to settle and separated. The aqueous layer isrewashed twice with 2.2 L dichloromethane. The combined organic layersare washed with a solution of 625 g ammonium chloride in 2.25 L water.(The ammonium chloride buffers the pH of the aqueous extractions to bewithin a range of from about pH 1-2 to about pH 4-5, as strongly acidicconditions might cleave peptide bonds). The organic phase is dried overmagnesium sulfate, filtered and the filtrate evaporated to dryness. Anassay of the free boronic acid is performed (by the RP HPLC method ofExample_for at most 30 mins (optionally about 20 min) at roomtemperature) and the amounts of the solvents and base for conversion ofthe acid to the salt are calculated. If 2.5 mol of the free acid isobtained, the evaporation residue is dissolved in 5 L acetonitrilefollowed by addition of a solution of 100 g (2.5 mole) sodium hydroxideas a 5% solution in 2.2 L water. The solution is stirred for two hoursat ambient temperature (e.g. 15-30° C., optimally room temperature) andthen evaporated in vacuum (of ca. 10 mmHg) at a temperature notexceeding 35° C. The evaporation residue is repeatedly dissolved in 3.5L fresh acetonitrile and evaporated to dryness to remove traces ofwater. If the evaporation residue is dry, it is dissolved in 3 Lacetonitrile (or alternatively in 6 L THF) and slowly added to a mixtureof 32 L n-heptane and 32 L diethylether. The addition is performedslowly enough to avoid lumping or sticking of the product and is carriedout over a period of not less than 30 minutes. The precipitated productis filtered off, washed with n-heptane and dried under vacuum at atemperature initially of about 10° C. and then increasing to a limit ofabout 35° C., until dry.

Yield: 1.0 kg (70%) Tri50c sodium salt.

Example 4 Preparation of Calcium Salt of TRI 50C

1.5 kg (2.5 mole) TRI 50d from Example 2 is dissolved in 10.5 Ldichloromethane. 11 L 2% hydrochloric acid is added and the mixture isstirred for at most 30 minutes (optimally about 20 minutes) at roomtemperature. After stirring the layers are allowed to settle andseparated. The aqueous layer is given a rewashed twice with 2.2 Ldichloromethane. The combined organic layers are washed with a solutionof 625 g ammonium chloride in 2.25 L water. The organic phase is driedover magnesium sulfate, filtered and the filtrate evaporated to dryness.An assay of the free boronic acid is performed and the amounts of thesolvents and base for conversion of the acid to the salt are calculated.If 2.5 mol of the free acid is obtained, the evaporation residue isdissolved in 5 L acetonitrile followed by addition of a suspension of 93g (1.25 mole) calcium hydroxide in 1 L water. The solution is stirredfor two hours at ambient temperature (e.g. 15-30° C., optimally roomtemperature) and then evaporated under vacuum (of ca. 10 mmHg) at atemperature initially of about 10° C. and then increasing to a limit ofabout 35° C. The evaporation residue is repeatedly dissolved in 3.5 Lfresh acetonitrile and evaporated to dryness to remove traces of water.If the evaporation residue is dry, it is dissolved in 6 Ltetrahydrofuran and slowly added to a mixture of 32 L n-heptane and 32 Ldiethylether. The addition is performed slowly enough to avoid lumpingor sticking of the product and is carried out over a period of not lessthan 30 minutes. The precipitated product is filtered off, washed withn-heptane and dried under vacuum (of ca. 10 mmHg) at a temperature below35° C. until dry.

Yield: 0.98 kg (70%) Tri50c calcium salt.

The procedures of Examples 1 to 4 may be scaled up and, if operatedcarefully, will produce highly pure salts. In the diethanolamineprecipitation step it is important to use 1.25 equivalents ofdiethanolamine per equivalent of (R,S,R) TRI 50b. In the hydrolysis ofthe diethanolamine ester, it is important to avoid excessively longcontact with the aqueous acid. Likewise the TRI 50b should besynthesised via the Grignard reaction to Z-DIPIN A.

Example 5 Alternative Conversion of TRI 50B TO TRI 50C

The synthetic procedures described in this and subsequent syntheticexamples were generally performed under nitrogen and using dry solventsas supplied from commercial sources.

1. Approximately 300 g of TRI 50b, obtained by the HPLC purification ofracemic TRI 50b) were dissolved in approximately 2.5 L diethylether. Itis estimated that different batches of TRI 50b had Isomeric puritiesranging from 85% R,S,R to in excess of 95% R,S,R.2. Approximately 54 ml diethanolamine were added (1:1 stoichiometry withtotal TRI 50b content), and the mixture was refluxed at 40° C.3. The precipitated product was removed, washed several times withdiethylether and dried.4. The dry product was dissolved in CHCl₃. Hydrochloric acid (pH 1) wasadded and the mixture was stirred approximately 1 h at room temperature.5. The organic layer was removed and washed with NH₄Cl solution.6. The organic solvent was distilled off and the residual solid productwas dried.

Typical yield: Approximately 230 g

Example 6 Preparation of Lithium Salt of TRI 50C

Cbz-Phe-Pro-BoroMpg-OH obtained by the method of Example 5 (20.00 g,38.1 mM) is dissolved in acetonitrile (200 ml) with stirring at roomtemperature. To this solution is added LiOH as a 0.2M solution indistilled water (190 ml). The resultant clear solution is stirred for 2hours at room temperature and then evacuated to dryness under vacuumwith its temperature not exceeding 37° C. The resultant oil/tacky liquidis redissolved in 500 ml distilled water necessary with light warmingfor about 20 minutes. The solution is filtered through filter paper andevacuated to dryness, again with the temperature of the solution notexceeding 37° C. The resultant product is dried under vacuum overnightto normally yield a white brittle solid.

The salt was then dried under vacuum over silica to constant weight (72h).

Yield 17.89 g.

Microanalysis:

C % Found H % Found N % Found B % Found Metal % Found (Calc.) (Calc.)(Calc.) (Calc.) (Calc.) 57.14 6.60 7.34 2.07 Li 1.26 (61.03) (6.64)(7.90) (2.03) (1.31)

Example 7 UV/Visible Spectra Of Lithium Salt of TRI 50C

UV/Visible spectra of the salt resulting from the procedure of Example 6were recorded in distilled water at 20° C. from 190 nm to 400 nm. Thesalt gave λ_(max) at 210 and 258 nm. The weight of the dried salt wasthen measured for the purposes of calculating the extinctioncoefficient. The λ_(max) at 258 nm was used. The extinction coefficientwas calculated using the formula:—

A=εcl

-   -   where A is the absorbance    -   C is the concentration    -   I the path length of the UV cell    -   and ε is the extinction coefficient

Extinction coefficient: 451

Example 8 Aqueous Solubility of Lithium Salt of TRI 50C

The salt used in this Example was made using a modification of theprocess described in Example 6. The modified process differs from thatdescribed in that 100 mg of TRI 50c was used as starting material, theproduct of the redissolution in water was dried by freeze drying and thefiltration was carried out through a 0.2 μm filter. The salt is believedto contain about 85% of R,S,R Isomer.

To determine maximum aqueous solubility 25 mg of the dried salt wereshaken in water at 37° C., the sample filtered and the UV spectrummeasured. The salt left a white residue of undissolved material. Thelithium salt was comparatively soluble and so was redissolved at 50mg/ml in the same manner previously described.

Solubility when dissolved at 25 mg/ml: 43 mM (23 mg/ml).

Solubility when dissolved at S0 mg/ml: 81 mM (43 mg/ml).

Example 9 Preparation of Sodium Salt of TRI 50C

Cbz-Phe-Pro-BoroMpg-OH obtained by the method of Example 5 (20.00 g,38.1 mM) is dissolved in acetonitrile (200 ml) with stirring at roomtemperature. To this solution is added NaOH as a 0.2M solution indistilled water (190 ml). The resultant clear solution is stirred for 2hours at room temperature and then evacuated to dryness under vacuumwith its temperature not exceeding 37° C. The resultant oil/tacky liquidis redissolved in 500 ml distilled water with light warming for about15-20 minutes. The solution is filtered through filter paper andevacuated to dryness, again with the temperature of the solution notexceeding 37° C. The resultant product is dried under vacuum overnightto normally yield a white brittle solid. The product may be present asan oil or tacky solid due to residual water, in which case it isdissolved in ethyl acetate and evacuated to dryness to produce theproduct as a white solid.

The salt was then dried under vacuum over silica to constant weight (72h).

Yield: Over 50%.

Microanalysis:

C % Found H % Found N % Found B % Found Metal % Found (Calc.) (Calc.)(Calc.) (Calc.) (Calc.) 59.93 6.47 7.31 1.91 Na 3.81 (59.24) (6.44)(7.67) (1.98) (4.20)

Example 10 UV/Visible Spectra of Sodium Salt of TRI50C

UV/Visible spectra of the sodium salt resulting from the procedure ofExample 9 were recorded in distilled water at 20° C. from 190 nm to 400nm. The salt gave λ_(max) at 210 and 258 nm. The weight of the driedsalt was then measured for the purposes of calculating the extinctioncoefficient. The λ_(max) at 258 nm was used. The extinction coefficientwas calculated using the formula:—

A=εcl

-   -   where A is the absorbance    -   C is the concentration    -   I the path length of the UV cell    -   and ε is the extinction coefficient.

Extinction coefficient: 415.

Example 11 Aqueous Solubility of Sodium Salt of TRI50C

The salt used in this Example was made using a modification of theprocess described in Example 9. The modified process differs from thatdescribed in that 100ng of TRI 50c was used as starting material, theproduct of the redissolution in water was dried by freeze drying and thefiltration was carried out through a 0.2 μm filter. The salt is believedto contain about 85% of R,S,R isomer. To determine maximum aqueoussolubility 25 mg of the dried salt were shaken in water at 37° C., thesample filtered and the UV spectrum measured. The salt left a whiteresidue of undissolved material. The sodium salt was comparativelysoluble and so was redissolved at 50 mg/ml in the same manner previouslydescribed.

Solubility when dissolved at 25 mg/ml: 44 mM (25 mg/ml).

Solubility when dissolved at 50 mg/ml: 90 mM (50 mg/ml).

Example 12 Preparation of Potassium Salt of TRI50C

Cbz-Phe-Pro-BoroMpg-OH obtained by the method of Example 5 (20.00 g,38.1 mM) is dissolved in acetonitrile (200 ml) with stirring at roomtemperature. To this solution is added KOH as a 0.2M solution indistilled water (190 ml). The resultant clear solution is stirred for 2hours at room temperature and then evacuated to dryness under vacuumwith its temperature not exceeding 37° C. The resultant oil/tacky liquidis redissolved in 1 L distilled water with warming to 37° C. for about 2hours. The solution is filtered through filter paper and evacuated todryness, again with the temperature of the solution not exceeding 37° C.The resultant product is dried under vacuum overnight to normally yielda white brittle solid.

Yield: 14.45 mg.

The salt was then dried under vacuum over silica to constant weight (72h).

Microanalysis:

C % Found H % Found N % Found B % Found Metal % Found (Calc.) (Calc.)(Calc.) (Calc.) (Calc.) 54.84 6.25 7.02 2.01 K 4.29 (57.55) (6.26)(7.45) (1.92) (6.94)

Example 13 UV/Visible Spectra of Potassium Salt of TRI50C

UV/Visible spectra of the potassium salt resulting from the procedure ofExample 12 were recorded in distilled water at 20° C. from 190 nm to 400nm. TRI50C and the salt gave Relax at 210 and 258 nm. The weight of thedried salt was then measured for the purposes of calculating theextinction coefficient. The λ_(max) at 258 nm was used. The extinctioncoefficient was calculated using the formula:—

A=εcl

-   -   where A is the absorbance    -   C is the concentration    -   I the path length of the UV cell    -   and ε is the extinction coefficient.

Extinction coefficient: 438.

Example 14 Aqueous Solubility of Potassium Salt of TRI50C

The salt used in this Example was made using a modification of theprocess described in Example 12. The modified process differs from thatdescribed in that 100 mg of TRI 50c was used as starting material, theproduct of the redissolution in water was dried by freeze drying and thefiltration was carried out through a 0.2 μm filter. The salt is believedto contain about 85% of R,S,R isomer.

To determine maximum aqueous solubility 25 mg of the dried salt wereshaken in water at 37° C., the sample filtered and the UV spectrummeasured. The salt left a white residue of undissolved material.

Solubility when dissolved at 25 mg/ml: 29 mM (16 mg/ml).

Example 15 Preparation of Zinc Salt of TRI 50C

The relative solubility of zinc hydroxide is such that, if the hydroxidehad been used to prepare the corresponding TRI 50c salt using theprocedure of Example 6, they would not have resulted in homogeneous saltformation. A new method was therefore developed to prepare the zincsalt, as described in this and the next examples.

TRI 50c sodium salt (2.24 g, 4.1 mM) was dissolved in distilled water(100 ml) at room temperature and zinc chloride in THF (4.27 ml, 0.5M)was carefully added with stirring. A white precipitate that immediatelyformed was filtered off and washed with distilled water. This solid wasdissolved in ethyl acetate and washed with distilled water (2×50 ml).The organic solution was evacuated to dryness and the white solidproduced dried over silica in a desiccator for 3 days beforemicroanalysis. Yield 1.20 g.

¹H NMR 400 MHz, δ_(H)(CD₃OD) 7.23-7.33 (20H, m, ArH), 5.14 (4H, m,PhCH₂O), 4.52 (4H, m, αCH), 3.65 (2H, m), 3.31 (12H, m), 3.23 (6H, s,OCH₃), 2.96 (4H, d, J7.8 Hz), 2.78 (2H, m), 2.58 (2H, m), 1.86 (6H, m),1.40 (10H, m).

¹³C NMR 75 MHz δ_(C)(CD₃OD) 178.50, 159.00, 138.05, 137.66, 130.54,129.62, 129.50, 129.07, 128.79, 128.22, 73.90, 67.90, 58.64, 58.18,56.02, 38.81, 30.06, 28.57, 28.36, 25.29. FTIR (KBr disc) ν_(max) (cm⁻¹)3291.1, 3062.7, 3031.1, 2932.9, 2875.7, 2346.0, 1956.2, 1711.8, 1647.6,1536.0, 1498.2, 1452.1, 1392.4, 1343.1, 1253.8, 1116.8, 1084.3, 1027.7,916.0, 887.6, 748.6, 699.4, 595.5, 506.5.

Example 16 Preparation of Arginine Salt of TRI50C

Cbz-Phe-Pro-BoroMpg-OH obtained by the method of Example 5 (20.00 g,38.1 mM) is dissolved in acetonitrile (200 ml) with stirring at roomtemperature. To this solution is added arginine as a 0.2M solution indistilled water (190 ml). The resultant clear solution is stirred for 2hours at room temperature and then evacuated to dryness under vacuumwith its temperature not exceeding 37° C. The resultant oil/tacky liquidis redissolved in 2 L distilled water with warming to 37° C. for 2hours. The solution is filtered through filter paper and evacuated todryness, again with the temperature of the solution not exceeding 37° C.The resultant product is dried under vacuum overnight to normally yielda white brittle solid.

The salt was then dried under vacuum over silica to constant weight (72h).

Yield: 10.54 g.

Microanalysis:

C % Found H % Found N % Found B % Found (Calc.) (Calc.) (Calc.) (Calc.)52.47 7.12 15.25 1.52 (56.65) (7.20) (14.01) (1.54)

Example 17 UV/Visible Spectra of Arginine Salt of TRI50C

UV/Visible spectra of the salt resulting from the procedure of Example15 were recorded in distilled water at 20° C. from 190 nm to 400 nm.TRI50C and the salt gave λ_(max) at 210 and 258 nm. The weight of thedried salt was then measured for the purposes of calculating theextinction coefficient. The λ_(max) at 258 nm was used. The extinctioncoefficient was calculated using the formula:—

A=εcl

-   -   where A is the absorbance    -   C is the concentration    -   I the path length of the UV cell    -   and ε is the extinction coefficient.

Extinction coefficient: 406.

Example 18 Aqueous Solubility of Arginine Salt of TRI50C

The salt used in this Example was made using a modification of theprocess described in Example 16. The modified process differs from thatdescribed in that 100 mg of TRI 50c was used as starting material, theproduct of the redissolution in water was dried by freeze drying and thefiltration was carried out through a 0.2 μm filter. The salt is believedto contain about 85% of R,S,R isomer.

To determine maximum aqueous solubility 25 mg of the dried salt wereshaken in water at 37° C., the sample filtered and the UV spectrummeasured. The salt left a white residue of undissolved material.

Solubility when dissolved at 25 mg/ml: 14 mM (10 mg/ml).

Example 19 Preparation of Lysine Salt of TRI50C

Cbz-Phe-Pro-BoroMpg-OH obtained by the method of Example 5 (20.00 g,38.1 mM) is dissolved in acetonitrile (200 ml) with stirring at roomtemperature. To this solution is added L-lysine as a 0.2M solution indistilled water (190 ml). The resultant clear solution is stirred for 2hours at room temperature and then evacuated to dryness under vacuumwith its temperature not exceeding 37° C. The resultant oil/tacky liquidis redissolved in 3 L distilled water with warming to 37° C. for 2hours. The solution is filtered through filter paper and evacuated todryness, again with the temperature of the solution not exceeding 37° C.The resultant product is dried under vacuum overnight to normally yielda white brittle solid. The product may be present as an oil or tackysolid (due to residual water), in which case it is then dissolved inethyl acetate and evacuated to dryness to produce the product as a whitesolid.

The salt was then dried under vacuum over silica to constant weight (72h).

Yield: 17.89.

Microanalysis:

C % Found H % Found N % Found B % Found (Calc.) (Calc.) (Calc.) (Calc.)57.03 7.43 10.50 1.72 (59.11) (7.36) (10.44) (1.61)

Example 20 UV/Visible Spectra of Lysine Salt of TRI50C

UV/Visible spectra of the salt resulting from the procedure of Example19 were recorded in distilled water at 20° C. from 190 nm to 400 nm.TRI50C and the salt gave λ_(max) at 210 and 258 nm. The weight of thedried salt was then measured for the purposes of calculating theextinction coefficient. The λ_(max) at 258 nm was used. The extinctioncoefficient was calculated using the formula:—

A=εcl

-   -   where A is the absorbance    -   C is the concentration    -   I the path length of the UV cell    -   and ε is the extinction coefficient.

Extinction coefficient: 437.

Example 21 Aqueous Solubility of Lysine Salt of TRI50C

The salt used in this Example was made using a modification of theprocess described in Example 19. The modified process differs from thatdescribed in that 100 mg of TRI 50c was used as starting material, theproduct of the redissolution in water was dried by freeze drying and thefiltration was carried out through a 0.2 μm filter. The salt is believedto contain about 85% of R,S,R Isomer.

To determine maximum aqueous solubility 25 mg of the dried salt wereshaken in water at 37° C., the sample filtered and the UV spectrummeasured. The salt left a white residue of undissolved material.

Solubility when dissolved at 25 mg/ml: 13 mM (8.6 mg/ml).

Example 22 Preparation of N-Methyl-D-Glucamine Salt of TRI50C

Cbz-Phe-Pro-BoroMpg-OH obtained by the method of Example 5 (20.00 g,38.1 mM) is dissolved in acetonitrile (200 ml) with stirring at roomtemperature. To this solution is added N-methyl-D-glucamine as a 0.2Msolution in distilled water (190 ml). The resultant clear solution isstirred for 2 hours at room temperature and then evacuated to drynessunder vacuum with its temperature not exceeding 37° C. The resultantoil/tacky liquid is redissolved in 500 ml distilled water with lightwarming for about 20 minutes. The solution is filtered through filerpaper and evacuated to dryness, again with the temperature of thesolution not exceeding 37° C., or freeze dried. The resultant product isdried under vacuum overnight to normally yield a white brittle solid.

The salt was then dried under vacuum over silica to constant weight (72h)

Yield: 21.31 g.

Microanalysis:

C % Found H % Found N % Found B % Found (Calc.) (Calc.) (Calc.) (Calc.)56.67 7.28 7.74 1.63 (56.67) (7.41) (7.77) (1.50)

Example 23 UV/Visible Spectra of N-Methyl-D-Glucamine Salt of TRI50C

UV/Visible spectra of the salt resulting from the procedure of Example22 were recorded in distilled water at 20° C. from 19 nm to 400 nm.TRI50C and the salt gave λ_(max) at 210 and 258 nm. The weight of thedried salt was then measured for the purposes of calculating theextinction coefficient. The λ_(max) at 258 nm was used. The extinctioncoefficient was calculated using the formula:—

A=εcl

-   -   where A is the absorbance    -   C is the concentration    -   I the path length of the UV cell    -   and ε is the extinction coefficient.

Extinction coefficient: 433.

Example 24 Aqueous Solubility of N-Methyl-D-Glucamine Salt of TRI50C

The salt used in this Example was made using a modification of theprocess described in Example 22. The modified process differs from thatdescribed in that 100 mg of TRI 50c was used as starting material, theproduct of the redissolution in water was dried by freeze drying and thefiltration was carried out through a 0.2 μm filter. The salt is believedto contain about 85% of R,S,R isomer.

To determine maximum aqueous solubility 25 mg of the dried salt wereshaken in water at 37° C., the sample filtered and the UV spectrummeasured. The salt was observed to fully dissolve. The salt wascomparatively soluble and so was redissolved at 50 mg/ml in the samemanner previously described.

Solubility when dissolved at 25 mg/ml: 35 mM (25 mg/ml).

Solubility when dissolved at 50 mg/ml: 70 mM (50 mg/ml).

Example 25 Alternative Preparation of Arginine Salt of TRI50C

The arginine salt is formed simply by adding a slight molar excess ofL-arginine to a solution of 0.2-0.3 mmol of TRI50c in 10 ml of ethylacetate. The solvent is evaporated after one hour, and the residue istriturated twice with hexane to remove excess arginine.

Example 26 First Preparation of Calcium Salt of TRI 50C

Cbz-Phe-Pro-BoroMpg-OH (20.00 g, 38.1 mM) obtained by the method ofExample 5 is dissolved in acetonitrile (200 ml) with stirring at roomtemperature. To this solution is added Ca(OH)₂ as a 0.1M solution indistilled water (190 ml). The resultant clear solution is stirred for 2hours at room temperature and then evacuated to dryness under vacuumwith its temperature not exceeding 37° C. The resultant product is awhite brittle solid.

The salt was then dried under vacuum over silica to constant weight (72h).

Yield: 17.69 g.

Example 27 Second Alternative Preparation of Calcium Salt of TRI 50C

50.0 g TRI 50c (95.2 mmol) were dissolved under stirring in 250 nilacetonitrile at room temperature and then cooled with an ice bath. Tothis ice cooled solution 100 ml of an aqueous suspension of 3.5 g (47.6mmol) calcium hydroxide was added dropwise, stirred for 2.5 hours atroom temperature, filtered and the resulting mixture evaporated todryness, the temperature not exceeding 35° C. The clear yellowish oilyresidue was redissolved in 200 ml acetone and evaporated to dryness. Theprocedure of redissolving in acetone was repeated one more time toobtain colourless foam.

This foam was redissolved in 100 ml acetone, filtered and added dropwiseto an ice cooled solution of 1100 ml petrol ether 40/60 and 1100 mldiethylether. The resulting colourless precipitate was filtered, washedtwo times with petrol ether 40/60 and dried under high vacuum, yielding49.48 g of a colourless solid (92%), with a purity of 99.4% according toan HPLC measurement.

Example 28 UV/Visible Spectra of Calcium Salt of TRI 50C

UV/Visible spectra of the salt resulting from the procedure of Example26 were recorded in distilled water at 20° C. from 190 nm to 400 nm. TRI50C and the salt gave λ_(max) at 210 and 258 nm. The weight of the driedsalt was then measured for the purposes of calculating the extinctioncoefficient. The λ_(max) at 258 nm was used. The extinction coefficientwas calculated using the formula:—

A=εcl

-   -   where A is the absorbance    -   c is the concentration    -   I the path length of the UV cell    -   and ε is the extinction coefficient.

Extinction coefficient: 955.

Example 29 Aqueous Solubility of Calcium Salt of TRI 50C

The salt used in this Example was made using a modification of theprocess described in Example 27. The modified process differs from thatdescribed in that 100 mg of TRI 50c was used as starting material, theproduct of the redissolution in water was dried by freeze drying and thefiltration was carried out through a 0.2 μm filter. The salt is believedto contain about 85% of R,S,R Isomer.

To determine maximum aqueous solubility 25 mg of the dried salt wereshaken in water at 37° C., the sample filtered and the UV spectrummeasured. The salt left a white residue of undissolved material.

Solubility when dissolved at 25 mg/ml: 5 mM (5 mg/ml).

Example 30 In Vitro Activity of Calcium Salt of TRI 50C

TRI 50c calcium salt was assayed as an inhibitor of human α-thrombin byan amidolytic assay (J. Deadman et al, J. Med. Chem., 38:15111-1522,1995, which reports a Ki value of 7 nM for TRI 50b).

The inhibition of human α-thrombin therefore, was determined by theinhibition of the enzyme catalysed hydrolysis of three differentconcentrations of the chromogenic substrate S-2238.

200 μl of sample or buffer and 50 μl of S-2238 were incubated at 37° C.for 1 minute and 50 μl of human α-thrombin (0.25 NIH_(μ)/ml) was added.The initial rate of Inhibited and uninhibited reactions were recorded at4.5 nm. The increase in optical density was plotted according to themethod of Lineweaver and Burke. The Km and apparent Km were determinedand Ki was calculated using the relationship.

$V = \frac{V\; \max}{1 + {\frac{Km}{\lbrack S\rbrack} \cdot \left( {1 + \frac{\lbrack I\rbrack}{Ki}} \right)}}$

The buffer used contained 0.1M sodium phosphate, 0.2M NaCl, 0.5% PEG and0.02% sodium azide, adjusted to pH 7.5 with orthophosphoric add.

The samples consist of the compound dissolved in DMSO.

The reader is referred to Dixon, M. and Webb, E. C., “Enzymes”, thirdedition, 1979, Academic Press, the disclosure of which is incorporatedherein by reference, for a further description of the measurement of Ki.

TRI 50c calcium salt was observed to have a Ki of 10 nM.

Example 31 Preparation of Magnesium Salt of TRI 50C

TRI 50c (1.00 g, 1.90 mM) was dissolved in methanol (10 ml) and stirredat room temperature. To this solution was added magnesium methoxide(Mg(CH₃O)₂) in methanol (1.05 ml, 7.84 wt %). This solution was stirredfor 2 hours at room temperature filtered and evacuated to 5 ml. Water(25 ml) was then added and the solution evacuated down to dryness toyield a white solid. This was dried over silica for 72 hours beforebeing sent for microanalysis. Yield 760 mg.

¹H NMR 300 MHz, δ_(H)(CD₃C(O)CD₃) 7.14-7.22 (2H, m), 6.90 (2H, m), 4.89(4H, m, PhCH₂O), 4.38 (2H, m), 3.40 (2H, br s), 2.73-3.17 (2H, broadunresolved multiplets), 1.05-2.10 (16H, broad unresolved multiplets).

¹³C NMR 75 MHz δ_(C)(CD₃C(O)CD₃) 206.56, 138.30, 130.76, 129.64, 129.31,129.19, 129.09, 128.20, 128.04, 74.23, 73.55, 67.78, 58.76, 56.37,56.03, 48.38, 47.87, 39.00, 25.42, 25.29. FTIR (KBr disc) λ_(max) (cm⁻¹)3331.3, 3031.4, 2935.3, 2876.9, 2341.9, 1956.1, 1711.6, 1639.9, 1534.3,1498.1, 1453.0, 1255.3, 1115.3, 1084.6, 1027.6, 917.3, 748.9, 699.6,594.9, 504.5, 467.8.

Example 32 Solubility of TRI50C

The UV/visible spectra of TRI50c resulting from the procedure of Example5 and Its solubility were obtained as described above in relation to thesalts. The solubility of TRI 50c when dissolved at 50 mg/ml was 8 mM (4mg/ml).

Example 33 Analysis of Sodium, Calcium, Magnesium and Zinc Salts of(R,S,R TRI 50C

The following salts were prepared using a boronate:metal stoichiometryof n:1, where n is the valency of the metal, using (R,S,R) TRI 50c ofhigher chiral purity than that used to prepare the salts described inExamples 8, 11, 14, 18, 21, 24 and 29.

A. Sodium Salt (Product of Example 9) Analytical data HPLC or LC/MS:HPLC betabasic C18 Column, CH₃CN, Water Estimated Purity: >95% by UV(λ_(215 nm)) Micro analysis: Calcd. Found. C: 59.24 59.93 H: 6.44 6.47N: 7.67 7.31 Other: B: 1.98 1.91 Na: 4.20 3.81 Physical Properties Form:Amorphous solid Colour: White Melting Point: N/A Solubility: Soluble inaqueous media ca ~50 mg/ml M_(w): 547.40 B. Calcium Salt (Product ofExample 26) Analytical data HPLC or LC/MS: HPLC betabasic C18 Column,CH₃CN, Water Estimated Purity: >95% by UV (λ_(215 nm)) Micro analysis:Calcd. Found. C: 59.27 55.08 H: 6.48 6.43 N: 7.71 7.08 Other: B: 1.992.01 Ca: 3.68 3.65 Physical Properties Form: Amorphous solid Colour:White Melting Point: N/A Solubility: Soluble in aqueous media ca ~4mg/ml M_(w): 1088.89 C. Magnesium Salt (Product of Example 31)Analytical data HPLC or LC/MS: HPLC betabasic C18 Column, CH₃CN, WaterEstimated Purity: >90% by UV (λ_(215 nm)) Micro analysis: Calcd. Found.C: 60.44 57.25 H: 6.57 6.71 N: 7.83 7.45 Other: B: 2.01 2.02 Mg: 2.262.12 Physical Properties Form: Amorphous solid Colour: White MeltingPoint: N/A Solubility: Soluble in aqueous media ca ~7 mg/ml M_(w):1073.12 D. Zinc Salt (Product of Example 15) Analytical data HPLC orLC/MS: HPLC betabasic C18 Column, CH₃CN, Water Estimated Purity: >95% byUV (λ_(215 nm)) Micro analysis: Calcd. Found. C: 58.21 56.20 H: 6.336.33 N: 7.54 7.18 Other: B: 1.94 1.84 Zn: 5.87 7.26 Physical PropertiesForm: Amorphous solid Colour: White Melting Point: N/A Solubility:Soluble in aqueous media ca ~2 mg/ml M_(w): 1114.18 Notes: The trigonalformula of the acid boronate is used in the calculated microanalyses. Itis believed that a lower sodium salt solubility is reported in example11 because the salt tested in example 11 had lower chiral purity.

Conclusion

The zinc, calcium and magnesium salts have all been prepared with astoichiometry of one metal ion to two molecules of TRI 50c. The valuesfound for the calcium and magnesium salts are close to and thusconsistent with those calculated for this 1:2 stoichiometry. For thezinc salt an excess of zinc was found; nonetheless, the zinc saltcomprises a significant proportion of acid boronate. The sodium salt hasbeen prepared with a stoichiometry of one metal ion to one molecule ofTRI 50c. The value found for the sodium salt is close to and thusconsistent with that calculated for this 11 stoichiometry.

Example 34 Stability

An assay of TRI150c and its sodium and lysine salts before and afterdrying.

1. Tabulated Results

TABLE 1 Compound Amount [μg/mL] Purity (% area) TRI 50c dry 1000.0 82.00TRI 50c non-dried 947.3 85.54 TRI 50c Na salt dry 1024 98.81 TRI 50c Nasalt non-dried 1005.8 98.61 TRI 50c Lys salt dry 813.3 90.17 TRI 50c Lyssalt non-dried 809.8 92.25

The purity of the acid was lowered by the drying process but the purityof the salts was less affected; the purity of the sodium salt was notsignificantly reduced. Large differences in response factors will reducethe actual impurity levels, however.

2. Analytical Procedure 2.1 Sample Preparation

TRI 50c and its Na, U and Lys salts were weighed into HPLC vials andstored in a desiccator over phosphorus pentoxide for 1 week. For sampleanalysis, 5 mg of dried and non-dried material was weighed in a 5 mLvolumetric flask and dissolved in 1 mL acetonitrile and filled up withdemineralised water to 5 mL.

3. Data Evaluation

The quantitative evaluation was performed using an HPLC-PDA method.

4. Analytical Parameters 4.1 Equipment and Software

Autosampler Waters Alliance 2795 Pump Waters Alliance 2795 Column ovenWaters Alliance 2795 Detection Waters 996 diode array, MS-ZQ 2000 singlequad Software version Waters Millennium Release 4.0

4.2 Stationary Phase

Analytical Column ID S71 Material X-Terra ™ MS C₁₈, 5 μm SupplierWaters, Eschborn, Germany Dimensions 150 mm × 2.1 mm (length, internaldiameter)

4.3 Mobile Phase

Aqueous phase: A: H₂O + 0.1% Organic phase: C: ACN Gradient conditions:Time Flow % A % C  0.00 0.5 90 10 27.0 0.5 10 90 27.1 0.5 90 10 30.0 0.590 10

This example indicates that the salts of the disclosure, particularlythe metal salts, e.g. alkali metal salts, are more stable than the adds,notably TRI 50c.

Example 35 In-Vitro Assay as Thrombin Inhibitor of Magnesium Salt of TRI50C

Thrombin Amidolytic Assay

TRI 50c magnesium salt (TRI 11405) was tested in a thrombin amidolyticassay.

Reagents:

Assay Buffer:

100 mM Na phosphate

200 mM NaCl (11.688 g/l)

0.5% PEG 6000 (5 g/l)

0.02% Na azide

pH 7.5

Chromogenic substrate 52238 dissolved to 4 mM (25 mg/ml) in water.Diluted to 50 uM with assay buffer for use in assay at 5_(μ)M. (S2238 isH-D-Phe-Pip-Arg-pNA).

Thrombin obtained from HTI, via Cambridge Bioscience, and aliquoted at 1mg/ml with assay buffer. Dilute to 10 ng/ml with assay buffer and then afurther 1 in 3 for use in the assay.

Assay:

1101 assay buffer50 μl 5 μg/ml thrombin20 μl vehicle or compound solution

min at 37° C. 20 μl 50 μM 52238

Read at 405 nm at 37° C. for 10 minutes and record λ_(max)

Results:

The results are presented in FIG. 1.

Discussion:

In this assay the magnesium salt of TRI 50c shows the same activity asTRI 50b as an external control.

Example 36 TRI 50B Inhibition of Platelet Procoagulant Activity

Platelet pro-coagulant activity may be observed as the increase, in rateof activation of prothrombin by factor Xa in the presence of factor Vaupon the addition of platelets pretreated with thrombin, caused bythrombin alone, collagen alone or a mixture of thrombin and collagen.This property is due to an increase in anionic phospholipid on thesurface of the platelet with concomitant release of microvesicle fromthe surface. This is an essential physiological reaction and peoplewhose platelets have reduced ability to generate procoagulant activity(Scott syndrome) show an increased tendency for bleeding.

Method:

Washed platelets were treated with either 115 nM thrombin, 23 μg/mlcollagen or a mixture of both at the same concentration at 37° C. TRI50b was added either for 1 minute prior to the addition of activator orimmediately after the incubation with activator. Platelet procoagulantactivity was determined as described previously (Goodwin C A et al,Biochem J. 1995 8, 308: 15-21).

TRI 50b proved to be a potent Inhibitor of platelet procoagulantactivity with IC₅₀'s as summarised below:

Table 2: Influence of TRI 50b on the induction of platelet procoagulantactivity by various agonists:

TABLE 2 IC50 plus pre- IC50 without Fold acceleration incubationincubation Agonist without TRI 50b (nM) (nM) Thrombin 30 8 3000 Collagen45 200 300 Thrombin/Collagen 110 3 80

Table 2 records, for example, that when platelets were treated withthrombin they caused a 30-fold acceleration of the rate of activation ofprothrombin in comparison with control platelets. Treatment with TRI 50reduced such acceleration by half at the various TRI 50 concentrationlevels given. The significant potency of TRI 50 is evidenced by the factthat the IC₅₀ values are in the nanomolar range.

TRI 50b does not have an effect on ADP, collagen or epinephrine inducedaggregation of washed platelets.

Example 37 Rabbit Extracorporeal Shunt Model Introduction

The technique describes an animal model in which a platelet richthrombus is produced. The activity of TRI 50b and heparin are compared.

The carotid artery and jugular vein of anaesthetised rabbits were usedto create an extracorporeal circuit containing a suspended foreignsurface (silk thread). Thrombus deposition Is initiated by creation ofhigh sheer stress turbulent arterial blood flow, platelet activation,followed by coagulation in the presence of thrombogenic surfaces.Histopathological studies have shown that the thrombus is platelet rich.

Materials and Methods Animals:

NZW rabbits (males 2.5-3.5 kg) were used. The animals were allowed foodand water up to the induction of anaesthesia.

Anaesthesia:

Animals were premedicated with fontanel/fluanisone (Hypnorm) 0.15 mltotal by intramuscular Injection. General anaesthesia was induced withmethohexitone (10 mg/ml) to effect, followed by endotracheal intubation.Anaesthesia was maintained with isoflurane (1-2.0%) carried inoxygen/nitrous oxide.

Surgical Preparation:

The animals were placed in dorsal recumbency and the ventral cervicalregion prepared for surgery. The left carotid artery and right jugularvein were exposed. The artery was cannulated with a large Portex®catheter (yellow gauge), cut to a suitable length. The vein wascannulated with a Silastic® catheter. The shunt comprised of a 5 cmlength of auto analyser line (purple/white gauge). Joins to the shunt onthe arterial side were made with intermediate size Silastic® tubing. Theshunt was filled with saline before exposure to the circulation. Theright femoral artery was cannulated for the measurement of bloodpressure.

Thread Preparation and Insertion:

The central section of the shunt contained a thread 3 centimetres inlength. This consisted of 000 gauge Gutterman sewing silk so as to givefour strands with a single knot at the end. (The knot section wasoutside the shunt).

Blood Flow

Blood flow velocity was determined by use of ‘Doppler’ probes (CrystalBiotech). A silastic probe was positioned over the carotid artery at thepoint of insertion of the arterial catheter. Flow was recorded on achart recorder using heat sensitive paper.

Results

TABLE 3 THROMBUS WEIGHT ANTITHROMBOTIC TREATMENT DOSE AFTER 20 minuterun ACTIVITY Control N/A 22.4 ± 2.2 mg (n = 5) TRI 50b 10 mg/kg iv 9.78± 1.9 mg (n = 5) Active 3.0 mg/kg iv 15.3 ± 2.2 mg (n = 5) ActiveHEPARIN 100 u/kg iv 22.9 ± 1.65 mg (n = 4) Inactive 300 u/kg iv 10.5 ±1.4 mg (n = 4) Active (Severe bleeding)

Discussion

Table 3 shows that, under high arterial shear conditions, a TRI 50b doseof 3 mg/kg to 10 mg/kg iv significantly inhibits thrombus formationwithout bleeding, whereas a heparin dose within the normal clinicalrange for treating venous thrombosis (100 u/kg iv heparin) wasineffective. The higher dose of heparin, though active, caused severebleeding. These results, which show TRI 50b effectively inhibitingarterial thrombosis without causing bleeding, are consistent with TRI50b inhibiting platelet procoagulant activity. In contrast, the thrombininhibitor heparin, when administered at an approximately equi-effectivedose (in terms of inhibition of arterial thrombosis), produced thesevere bleeding normal when thrombin inhibitors are used to treatarterial thrombosis.

Example 38 Comparison of Bleeding Times

The aim of the study was to compare the bleeding times of heparin withTRI 50b in a suitable model. It is accepted that heparin is a poorinhibitor of platelet procoagulant activity (J. Biol. Chem. 1978 Oct.10; 253(19):6908-16; Miletich J P, Jackson C M, Majerus PW1: J. Clin.Invest 1983 May; 71(5):1383-91).

Bleeding times were determined in a rat tail bleeding model followingintravenous administration of heparin and TRI 50b. The doses employedwere chosen on the basis of their efficacy in the rat Wessler anddynamic models and were as follows:

TRI 50b: 5 and 10 mg/kg Heparin: 100 units/kg

Materials and Methods Anaesthesia

Rats were anaesthetised with sodium pentabarbitone at 60 mg/kg (2.0ml/kg of 30 mg/ml solution by ip. injection). Supplemental anaestheticwas given ip. as required.

Surgical Preparation

A jugular vein was cannulated for the administration of test compound.The trachea was also cannulated with a suitable cannula and the animalsallowed to breathe “room air” spontaneously.

Compound Administration

These were given in the appropriate vehicle at 1.0 ml/kg intravenously.Heparin was administered in saline, whilst TRI 50b was dissolved inethanol, and then the resultant solution added to water for injection (1part ethanol to 5 parts water).

Technique

Two minutes following compound administration the distal 2 mm of theanimal's tail was sectioned with a new scalpel blade and the tallimmersed in warm saline (370C) contained in a standard ‘universal’container, so that the blood stream was clearly visible. The bleedingtime recording was started immediately following transection until thecessation of blood flow from the tip of the tail. A period of 30 secondswas allowed after the blood flow from the tall had stopped to ensurethat bleeding did riot recommence, if bleeding did start again therecording time was continued for up to a maximum of 45 minutes.

Results

Table 4 gives a summary of the bleeding results and shows the increasesabove base line values.

TABLE 4 Summary table of bleeding results Bleeding time min Treatment(±SEM^(†)) Saline  5.1 ± 0.6 Heparin 100 u/kg iv >40* TRI 50b 5 mg/kg iv11.3 ± 1.2 TRI 50b 10 mg/kg 30.4 ± 5.2 iv *Severe bleeding in allanimals, with no cessation after 40 minutes. ^(†)SEM = standard error ofthe mean

Discussion

The results show that TRI 50b was superior to heparin (produced lessbleeding) at all doses. It should be noted that when 100 u/kg heparin iscompared with 5 mg/kg TRI 50b, heparin-treated animals bled moreextensively than those receiving TRI 50b; it was previously established(Example 25) that heparin at a dose of 100 u/kg is a less effectiveinhibitor of arterial thrombosis than TRI 50b at a dose of 3.0 mg/kg.Heparin is primarily a thrombin inhibitor and a poor Inhibitor ofplatelet procoagulant activity; the results are therefore consistentwith TRI 50b exerting anti-coagulant activity by inhibition of plateletcoagulant activity in addition to thrombin Inhibiting activity.

Example 39 TRI 50B as a Prodrug for TRI 50C: Pharmacokinetics andAbsorption Materials and Methods Animals

Rats, body weight circa 250-300 g were used. The animals were fastedonly on the day of use for the iv stage.

TABLE 5 iv phase Treatment Dose mg/kg iv n TRI 50b 1.0 mg/kg 3 TRI 50c1.0 mg/kg 3

Dose

Formulation (TRI 50b/TRI 50c)

These were dosed in a formulation prepared as follows: 48 mg/ml of TRI50b is dissolved in ethanol: PEG 300 (2:3 vol:vol). Just beforeadministration, 5 volumes of this solution is mixed with 3 volumes of 5%kollidon 17 8F.

i.v. Phase

Both compounds were given at a dose of 1.0 mg/kg iv.

The compounds were dosed in a PEG/ethanol/kollidon formulation which wasprepared immediately before, as described immediately under the heading“Dose”: Stock 15.0 mg/ml, This was dosed at 1.33 ml/kg (equivalent to 30mg/kg).

Blood Sampling

A pre dose sample was taken followed by: 0, 2, 5, 10, 20, 30, 40, 60 and90 minutes post dose.

Plasma

This was obtained by centrifugation (3000 RPM for 10 min) and stored at−20° C. prior to analysis.

Results Pharmacokinetic Analysis

TABLE 6 i.v. pharmacokinetic data TRI 50b TRI 50c Elimination half life:minutes 35 minutes 36.6 minutes Area under curve 1.68 1.48 MeanResidence Time 46 minutes   45 minutes Clearance: ml/min/kg 10 11.3Volume Distribution Litres/kg 0.5 0.59 Max Plasma Concentration(observed) 2.24 2.35

The following results are represented in FIG. 2:

FIG. 2: Intravenous phase clearance and kinetics following a single doseof TRI 50b or its free acid (TRI 50c). The figure shows the observedassay data.

Conclusion

The i.v. kinetics were similar for both TRI 50b and TRI 50c. The dataare consistent with TRI 50b being rapidly hydrolysed in plasma to TRI50c and with TRI 50c being the active principle.

The results of examples 36 to 39 indicate that administration of TRI 50cas a salt will provide a way to treat arterial thrombosis and/or venousthrombosis.

Example 40 Intravenous Administration of TRI 50C Sodium Salt

The pharmacokinetics (PK) and pharmacodynamics (PD) of TRI 50c sodiumsalt were studied in beagle dogs following intravenous administration.

The PD was measured as thrombin time and APTT using an automatedcoagulometer. Plasma concentrations were measured using an LCMS/MSmethod.

TRI 50c monosodium salt (108.8 g) was dissolved in 0.9% sodium chloride(100 ml) and dosed i.v. at 1.0 mg/kg (1.0 ml/kg over 30 seconds). Bloodsamples were taken into 3.8% tri-sodium citrate (1+8) at pre dose, 2, 5,10, 20, 30, minutes post dose and then at 1, 2, 3, 4, 6, 8, 12 and 24hours post dose. Plasma was prepared by centrifugation and frozen atminus 20° C. pending analysis.

Results

The sodium salt was tolerated well with no adverse events for the totalduration of the study.

Male and female dogs responded similarly with a pharmacodynamic C max:at 2 minutes (thrombin time of 154 seconds raised from a base line of14.3 seconds). Thrombin time was 26 seconds at one hour post dose.

There was an exceptionally good therapeutic ratio between the APTT andthrombin clotting time in dogs receiving the sodium salt at a dose of1.0 mg/kg i.v. Thrombin dotting time was elevated 10.8 times above baseline (154.4 seconds from 14.3 seconds) two minutes following dosing,compared to only 1.3 times elevation in the APTT (19 seconds to 25seconds post dose).

Example 41 Human Clinical Studies

In human clinical volunteer studies with doses of up to 2.5 mg/kg i.v.(dosages which significantly prolong the thrombin clotting time), TRI50b had no effect on Simplate bleeding time (i.e. bleeding time measuredusing a Simplate® bleeding time device).

It will be appreciated from the foregoing that the disclosure providesboronic acid salts useful for pharmaceutical purposes and which featureone or more of the following attributes: (1) improved hydrolyticstability; (2) improved stability against deboronation; and (3), in anyevent, not suggested by the prior art.

The selection of active ingredient for a pharmaceutical composition is acomplex task, which requires consideration not only of biologicalproperties (including bioavailability) but also of physicochemicalproperties desirable for processing, formulation and storage.Bioavailabillty itself is dependent on various factors, often includingin vivo stability, salvation properties and absorption properties, eachin turn potentially dependent on multiple physical, chemical and/orbiological behaviours.

The present disclosure includes the subject matter of the followingparagraphs:

1. A parenteral pharmaceutical formulation comprising a pharmaceuticallyacceptable base addition salt of a boronic acid of formula (I):

wherein

Y comprises a hydrophobic moiety which, together with the aminoboronicacid residue —NHCH(R⁹)—B(OH)₂, has affinity for the substrate bindingsite of thrombin; and

R⁹ is a straight chain alkyl group interrupted by one or more etherlinkages and In which the total number of oxygen and carbon atoms is 3,4, 5 or 6 or R⁹ is —(CH₂)_(m)—W where m is from 2, 3, 4 or 5 and W is—OH or halogen (F, Cl, Br or I).

2. A formulation of paragraph 1 wherein R⁹ is an alkoxyalkyl group3. A formulation of paragraph 1 or paragraph 2 wherein YCO— comprises anamino acid which binds to the 52 subsite of thrombin, the amino acidbeing N-terminally linked to a moiety which binds the S3 subsite ofthrombin.4. A formulation of paragraph 1 or paragraph 2 wherein Y is anoptionally N-terminally protected dipeptide which binds to the 53 and S2binding sites of thrombin and the peptide linkages in the acid areoptionally and independently N-substituted by a C₁-C₁₃ hydrocarbyloptionally containing in-chain or in-ring nitrogen, oxygen or sulfur andoptionally substituted by a substituent selected from halo, hydroxy andtrifluoromethyl.5. A formulation of paragraph 4 wherein said dipeptide is N-terminallyprotected and all the peptide linkages in the acid are unsubstituted.6. A formulation of paragraph 4 or paragraph 5 wherein the S3-bindingamino acid residue is of R configuration, the S2-binding residue is of Sconfiguration, and the fragment —NHCH(R⁹)—B(OH) is of R configuration.7. A formulation of any of paragraphs 1 to 6 wherein the boronic acidhas a Ki for thrombin of about 100 nM or less.8. A formulation of paragraph 7 wherein the boronic acid has a Ki forthrombin of about 20 nM or less.9. A formulation in parenteral dosage form of a pharmaceuticallyacceptable base addition salt of a boronic acid of formula (II):

where:

X is H (to form NH₂) or an amino-protecting group;

aa¹ is an amino acid having a hydrocarbyl side chain containing no morethan 20 carbon atoms and comprising at least one cyclic group having upto 13 carbon atoms;

aa² is an imino acid having from 4 to 6 ring members;

R¹ is a group of the formula —(CH₂)_(S)-Z, where s is 2, 3 or 4 and Z is—OH, —OMe, —OEt or halogen (F, Cl, Br or I).

10. A formulation of paragraph 9 wherein aa¹ is selected from Phe, Dpaand wholly or partially hydrogenated analogues thereof.11. A formulation of paragraph 9 wherein aa¹ is selected from Dpa, Phe,Dcha and Cha,12. A formulation of any of paragraphs 9 to 11 wherein aa¹ is ofR-configuration.13. A formulation of paragraph 9 wherein aa¹ is (R)-Phe or (R)-Dpa14. A formulation of paragraph 9 wherein aa¹ is (R)-Phe.15. A formulation of any of paragraphs 9 to 15 wherein aa² is a residueof an imino acid of formula (IV)

where R¹¹ is —CH₂—, —CH₂—CH₂—, —S—CH₂—, —S—C(CH₃)₂— or —CH₂—CH₂—CH₂—,which group, when the ring is 5- or 6-membered, is optionallysubstituted at one or more —CH₂— groups by from 1 to 3 C₁-C₃ alkylgroups.

16. A formulation of paragraph 15 wherein aa² is of 5-configuration.17. A formulation of paragraph 15 wherein aa² is an (5)-proline residue.18. A formulation of paragraph 9, wherein aa¹-aa² is (R)-Phe-(S)—Pro.19. A formulation of any of paragraphs 9 to 18 wherein R¹ is2-bromoethyl, 2-chloroethyl, 2-methoxyethyl, 3-bromopropyl,3-chloropropyl or 3-methoxypropyl.20. A formulation of any of paragraphs 9 to 18 wherein R¹ is3-methoxypropyl.21. A formulation of any of paragraphs 9 to 20 where X isR⁶—(CH₂)_(p)—C(O)—, R⁶—(CH₂)_(p)—S(O)₂—, R⁶—(CH₂)_(p)—NH—C(O)— orR⁶—(CH₂)_(p)—O—C(O)— wherein p is 0, 1, 2, 3, 4, 5 or 6 and R⁶ is H or a5 to 13-membered cyclic group optionally substituted by 1, 2 or 3substituents selected from halogen, amino, nitro, hydroxy, a C₅-C₆cyclic group, C₁-C₄ alkyl and C₁-C₄ alkyl containing, and/or linked tothe cyclic group through, an in-chain O, the aforesaid alkyl groupsoptionally being substituted by a substituent selected from halogen,amino, nitro, hydroxy and a C₅-C₆ cyclic group.22. A formulation of paragraph 21 wherein said 5 to 13-membered cyclicgroup is aromatic or heteroaromatic.23. A formulation of paragraph 22 wherein said 5 to 13-membered cyclicgroup is phenyl or a 6-membered heteroaromatic group.24. A formulation of any of paragraphs 9 to 20 wherein X isR⁶—(CH₂)_(p)—C(O)— or R⁶—(CH₂)_(p)—O—C(O)— and p is 0 or 1.25. A formulation of any of paragraphs 9 to 20 wherein X isbenzyloxycarbonyl.26. A formulation of paragraph 9 wherein the boronic acid is of formula(VIII):

X—(R)-Phe-(S)—Pro-(R)-Mpg-B(OH)₂  (VIII).

27. A formulation of any of paragraphs 1 to 26 wherein the saltcomprises boronate ions derived from the boronic acid and monovalentcounter-ions.28. A formulation of any of paragraphs 1 to 26 which comprises a salt ofthe peptide boronic acid with an alkali metal or a strongly basicorganic nitrogen-containing compound.29. A formulation of paragraph 28 wherein the strongly basic organicnitrogen-containing compound is a guanidine, a guanidine analogue or anamine.30. A formulation of any of paragraphs 1 to 27 wherein the salt is asalt of the boronic acid with a metal.31. A formulation of any of paragraphs 1 to 26 which comprises a salt ofthe boronic acid with an alkali metal, an aminosugar, a guanidine or anamine of formula (XI):

where n is from 1 to 6, R² is H, carboxylate or derivatised carboxylate,R³ is H, C₁-C₄ alkyl or a residue of a natural or unnatural amino acid.32. A formulation of any of paragraphs 1 to 26 which comprises a salt ofthe boronic acid with a guanidine or with an amine of formula (IX):

where n is from 1 to 6, R² is H, carboxylate or derivatised carboxylate,R³ is H, C₁-C₄ alkyl or a residue of a natural or unnatural amino acid.33. A formulation of paragraph 32 which comprises a guanidine salt ofthe boronic acid.34. A formulation of paragraph 33 which comprises a salt of the boronicacid with L-arginine or an L-arginine analogue.35. A formulation of paragraph 34 wherein the L-arginine analogue isD-arginine, or the D- or L-isomers of homoarginine, agmatine[(4-aminobutyl) guanidine], NG-nitro-L-arginine methyl ester, or a2-amino pyrimidines.36. A formulation of paragraph 33 which comprises a salt of the boronicacid with a guanidine of formula (VII)

where n is from 1 to 6, R² is H, carboxylate or derivatised carboxylate,RW is H, C₁-C₄ alkyl or a residue of a natural or unnatural amino acid.37. A formulation of paragraph 36, wherein n is 2, 3 or 4.38. A formulation of paragraph 36 or paragraph 37 where the derivatisedcarboxylate forms a C₁-C₄ alkyl ester or amide.39. A formulation of any of paragraphs 36 to 38 wherein the compound offormula (VII) is of L-configuration.40. A formulation of paragraph 33 which comprises an L-arginine salt ofthe peptide boronic acid.41. A formulation of paragraph 32 which comprises a salt of the boronicadd with an amine of formula (IX).42. A formulation of paragraph 41, wherein n is 2, 3 or 4.

43. A formulation of paragraph 41 or paragraph 42 where the derivatisedcarboxylate forms a C₁-C₄ alkyl ester or amide

44. A formulation of any of paragraphs 41 to 43 wherein the amine offormula (IX) is of L-configuration.45. A formulation of paragraph 41 which comprises an L-lysine salt ofthe boronic acid.46. A formulation of any of paragraphs 1 to 26 which comprises an alkalimetal salt of the boronic acid.47. A formulation of paragraph 46 wherein the alkali metal is potassium.48. A formulation of paragraph 46 wherein the alkali metal is sodium.49. A formulation of paragraph 46 wherein the alkali metal is lithium.50. A formulation of any of paragraphs 1 to 26 which comprises anaminosugar salt of the boronic add.51. A formulation of paragraph 50 wherein the aminosugar is aring-opened sugar.52. A formulation of paragraph 51 wherein the aminosugar Is a glucamine.53. A formulation of paragraph 50 wherein the aminosugar is a cyclicaminosugar.54. A formulation of any of paragraphs 50 to 53 wherein the aminosugaris N-unsubstituted.55. A formulation of any of paragraphs 50 to 53 wherein the aminosugaris N-substituted by one or two substituents.56. A formulation of paragraph 55 wherein the or each substituent is ahydrocarbyl group.57. A formulation of paragraph 55 wherein the or each substituent isselected from the group consisting of alkyl and aryl moieties.58. A formulation of paragraph 57 wherein the or each substituent isselected from the group consisting of C₁, C₂, C₃, C₄, C₅, C₆, C₇ and C₈alkyl groups59. A formulation of any of paragraphs 55 to 58 wherein there is asingle N-substituent.60. A formulation of paragraph 50 wherein the glucamine isN-methyl-D-glucamine.61. A formulation of any of paragraphs 1 to 60 which comprises boronateions derived from the peptide boronic acid and has a stoichiometryconsistent with the boronate ions carrying a single negative charge.62. A formulation of any of paragraphs 1 to 60 wherein the salt consistsessentially of acid salt (that is, wherein one B—OH group remainsprotonated).63. A formulation of any of paragraphs 1 to 62 wherein the saltcomprises a bororiate ion derived from the peptide boronic acid and acounter-ion and wherein the salt consists essentially of a salt having asingle type of counter-ion.64. A product for use as a parenteral pharmaceutical, comprising a saltof any of paragraphs 1 to 63.65. A pharmaceutical formulation in parenteral dosage form comprising asalt of any of paragraphs 1 to 63 and a pharmaceutically acceptablediluent, excipient or carrier.66. A pharmaceutical formulation of paragraph 65 which is adapted forintravenous administration.

67. A pharmaceutical formulation of paragraph 65 which is adapted forsubcutaneous administration.

68. A method of inhibiting thrombin in the treatment of diseasecomprising parenterally administering to a mammal a therapeuticallyeffective amount of an active agent selected from the group consistingof a salt as defined in any of paragraphs 1 to 63.69. The use of a salt as defined in any of paragraphs 1 to 63 for themanufacture of a parenteral medicament for treating thrombosis.70. A method of treating venous and/or arterial thrombosis byprophylaxis or therapy, comprising parenterally administering to amammal suffering from, or at risk of suffering from, arterial thrombosisa therapeutically effective amount of a product selected form the saltsdefined any of paragraphs 1 to 63.71. A method of paragraph 70 wherein the disease is an acute coronarysyndrome.72. A method of paragraph 70 wherein the disease is acute myocardialinfarction.73. A method of paragraph 70 wherein the disease is a venousthromboembolic event, selected from the group consisting of deep veinthrombosis and pulmonary embolism.74. A method for preventing thrombosis in a haemodialysis circuit of apatient, comprising parenterally administering to the patient atherapeutically effective amount of a product selected from the saltsdefined any of paragraphs 1 to 63.75. A method for preventing a cardiovascular event in a patient with endstage renal disease, comprising parenterally administering to thepatient a therapeutically effective amount of a product selected fromthe salts defined any of paragraphs 1 to 63.76. A method for preventing venous thromboembolic events in a patientreceiving chemotherapy through an indwelling catheter, comprisingadministering to the patient a therapeutically effective amount of aproduct selected from the salts defined any of paragraphs 1 to 63.77. A method for preventing thromboembolic events in a patientundergoing a lower limb arterial reconstructive procedure, comprisingparenterally administering to the patient a therapeutically effectiveamount of a product selected from the salts defined any of paragraphs 1to 63.78. A method of inhibiting platelet procoagulant activity, comprisingparenterally administering to a mammal at risk of, or suffering from,arterial thrombosis a therapeutically effective amount of a productselected from the salts defined any of paragraphs 1 to 63.79. A method of paragraph 78 wherein the disease is an acute coronarysyndrome.80. A method of treating by way of therapy or prophylaxis an arterialdisease selected from acute coronary syndromes, cerebrovascularthrombosis, peripheral arterial occlusion and arterial thrombosisresulting from atrial fibrillation, valvular heart disease,arterio-venous shunts, indwelling catheters or coronary stents,comprising parenterally administering to a mammal a therapeuticallyeffective amount of a product selected from the salts defined any ofparagraphs 1 to 63.81. A method of paragraph 80 wherein the disease is an acute coronarysyndrome.82. The use of a salt of any of paragraphs 1 to 63 for the manufactureof a parenteral medicament for a treatment recited in any of paragraphs76 to 81.83. A parenteral pharmaceutical formulation comprising a combination of(i) a salt of any of paragraphs 1 to 63 and (ii) a furtherpharmaceutically active agent.84. A parenteral pharmaceutical formulation comprising a combination of(i) a salt of any of paragraphs 1 to 63 and (ii) another cardiovasculartreatment agent.85. A formulation of paragraph 84 wherein the other cardiovasculartreatment agent comprises a lipid-lowering drug, a fibrate, niacin, astatin, a CETP inhibitor, a bile acid sequestrant, an anti-oxidant, aIIb/IIIa antagonist, an aldosterone inhibitor, an A2 antagonist, an A3agonist, a beta-blocker, acetylsalicylic acid, a loop diuretic, an aceinhibitor, an antithrombotic agent with a different mechanism of action,an antiplatelet agent, a thromboxane receptor and/or synthetaseinhibitor, a fibrinogen receptor antagonist, a prostacyclin mimetic, aphosphodlesterase Inhibitor, an ADP-receptor (P₂ T) antagonist, athrombolytic, a cardioprotectant or a COX-2 inhibitor.86. The use of a salt of any of paragraphs 1 to 63 for the manufactureof a parenteral medicament for treating, for example preventing, acardiovascular disorder in co-administration with another cardiovasculartreatment agent.87. A method for recovering from ether solution an ester of a boronicacid as defined in any of paragraphs 1 to 26, comprising dissolvingdiethanolamine in the solution, allowing or causing a precipitate toform and recovering the precipitate.88. A method of paragraph 79 wherein the ester is a pinacol ester.89. The method of paragraph 79 or paragraph 80 which further comprisesconverting, suitably hydrolysing, the precipitated material into thefree organoboronic acid.90. The method of paragraph 89, wherein the conversion comprisescontacting the precipitated material with an aqueous acid or base.91. The method of paragraph 90, wherein the precipitated material iscontacted with a concentrated strong Inorganic acid.92. A method for making a boronic add as defined in any of paragraphs 1to 26, comprising converting a diolamine reaction product thereof to theacid, suitably hydrolysing the diolamine reaction product to form theacid.93. The method of paragraph 92, wherein the conversion is carried out asrecited in paragraph 82 or paragraph 83.94. The method of any of paragraphs 87 to 93, which further comprisesconverting the organoboronic acid to a salt thereof.95. The method of paragraph 94, wherein the salt is as defined in any ofparagraphs 2 to 63.96. The method of paragraph 94 or paragraph 95, which further comprisesformulating the salt into a pharmaceutical composition.97. A product obtainable by (having the characteristics of a productobtained by) reacting in diethylether solution a pinacol ester of acompound of Formula (VIII) as defined in paragraph 26 anddiethanolamine.98. A composition of matter comprising:

-   -   (i) a species of formula (XII)

wherein X is H or an amino protecting group, the boron atom isoptionally coordinated additionally with a nitrogen atom, and thevalency status of the terminal oxygens is open (they may be attached toa second covalent bond, be ionised as —O⁻, or have some other, forexample intermediate, status); and, in bonding association therewith

-   -   (ii) a species of formula (XIII)

wherein the valency status of the nitrogen atom and the two oxygen atomsis open.99. A composition of paragraph 98, wherein the terminal oxygen atoms ofthe species of formula (XII) and the oxygen atoms of the species offormula (XIII) are the same oxygen atoms, i.e. the species of formula(XIII) forms a diol ester with the species of formula (XII).100. The use of a boronic acid as defined in any of paragraphs 1 to 26as an intermediate to make a salt of any of paragraphs 1 to 63.101. A method of preparing a salt of any of paragraphs 1 to 63,comprising contacting a boronic acid as defined in any of paragraphs 1to 26 with a base capable of making such a salt.102. A peptide boronic acid of formula (II) as defined in any ofparagraphs 9 to 26 when of GLP or GMP quality, or when in compliancewith GLP (good laboratory practice) or GMP (good manufacturingpractice).103. A composition of matter which is sterile or acceptable forpharmaceutical use, or both, and comprises a peptide boronic acid offormula (II) as defined in any of paragraphs 9 to 26.104. A composition of matter of paragraph 103 which is in particulateform.105. A composition of paragraph 103 which is in the form of a liquid,solution or dispersion.106. An isolated compound which is a peptide boronic acid of formula(VIII):

X—(R)-Phe-(S)—Pro-(R)-Mpg-B(OH)₂  (VIII)

wherein X is H (to form NH₂) or an amino-protecting group.107. A compound of paragraph 106 wherein X is benzyloxycarbonyl.108. A particulate composition comprising a peptide boronic acid offormula (VIII) as defined in paragraph 106 or paragraph 107.109. A composition of paragraph 108 consisting predominantly of thepeptide boronic acid.110. A composition of paragraph 109 wherein the peptide boronic acidforms at least 75% by weight of the composition.111. A composition of paragraph 110 wherein the peptide boronic acidforms at least 85% by weight of the composition.112. A composition of paragraph 111 wherein the peptide boronic acidforms at least 95% by weight of the composition.113. A composition of any of paragraphs 108 to 112 which is sterile.114. A composition of any of paragraphs 108 to 113 wherein the peptideboronic acid is in finely divided form.115. A liquid composition consisting of, or consisting essentially of, apeptide boronic acid of formula (II) as defined in any of paragraphs 9to 26 and liquid vehicle in which it is dissolved or suspended.116. A liquid composition of paragraph 115 wherein the liquid vehicle isan aqueous medium, e.g. water.117. A liquid composition of paragraph 115 wherein the liquid vehicle isan alcohol, for example methanol, ethanol, isopropanol or anotherpropanol, another alkanol or a mixture of the aforegoing.118. A liquid composition of any of paragraphs 115 to 117 which issterile.119. A parenteral medicament comprising a salt of a boronic acid whichis a selective thrombin inhibitor and has a neutral aminoboronic acidresidue capable of binding to the thrombin SI subsite linked through apeptide linkage to a hydrophobic moiety capable of binding to thethrombin 52 and S3 subsites, the salt comprising a cation having avalency n and having an observed stoichiometry consistent with anotional stoichiometry (boronic acid:cation) of n:1.120. A medicament of paragraph 119 wherein the boronic acid has a Ki forthrombin of about 100 nM or less.121. A medicament of paragraph 119 wherein the boronic acid has a Ki forthrombin of about 20 nM or less.122. A parenteral medicament comprising a sodium salt ofCbz-(R)-Phe-(S)—Pro-(R)-Mpg-B(OH)₂.123. A method of stabilising an organoboronic acid, comprising providingit in the form of a salt thereof.124. A method of formulating an organoboronic acid drug to increase thestability of the drug species, comprising formulating the acid in theform of an acid salt thereof.125. A pharmaceutical product comprising a sealed container containingin the form of a finely divided solid, ready for reconstitution to forma liquid parenteral formulation, a therapeutically effective amount of aboronate salt which consists essentially of a single pharmaceuticallyacceptable base addition salt of a boronic acid formula (II):

where:

X is H (to form NH₂) or an amino-protecting group;

aa¹ is an amino acid of R-configuration having a hydrocarbyl side chaincontaining no more than 20 carbon atoms and comprising at least onecyclic group having up to 13 carbon atoms;

aa² is an imino acid of S-configuration having from 4 to 6 ring members;

C* is a chiral centre of R-configuration; and

R¹ is a group of the formula —CH₂)_(s)-Z, where s is 2, 3 or 4 and Z is—OH, —OMe, —OEt or halogen (F, Cl, Br or I).

126. The product of paragraph 125 wherein:

X is R⁶—(CH₂)_(p)—C(O)—, R⁶—(CH₂)_(p)—S(O)₂—, R⁶—(CH₂)_(p)—NH—C(O)— orR⁶—(CH₂)_(p)—O—C(O)— wherein p is 0, 1, 2, 3, 4, 5 or 6 and R⁶ is H or a5 to 13-membered cyclic group optionally substituted by 1, 2 or 3substituents selected from halogen, amino, nitro, hydroxy, a C₅-C₆cyclic group, C₁-C₄ alkyl and C₁-C₄ alkyl containing, and/or linked tothe cyclic group through, an in-chain O, the aforesaid alkyl groupsoptionally being substituted by a substituent selected from halogen,amino, nitro, hydroxy and a C₅-C₆ cyclic group;

aa¹ is selected from (R)-Phe, (R)-Dpa, (R)—Cha and (R)-Dcha;aa² is Pro; andR¹ is 2-ethoxyethyl or 3-methoxypropyl.127. A pharmaceutical formulation adapted for parenteral administration,whether directly or after combining with a liquid, and comprising

-   -   a) a first species selected from (a) boronic acids of formula        (I), (b) boronate anions thereof, and (c) any equilibrium form        of the aforegoing (e.g. an anhydride):

wherein

Y comprises a hydrophobic moiety which, together with the aminoboronicacid residue —NHCH(R⁹)—B(OH)₂, has affinity for the substrate bindingsite of thrombin; and

-   -   R⁹ is a straight chain alkyl group interrupted by one or more        ether linkages and in which the total number of oxygen and        carbon atoms is 3, 4, 5 or 6 or R⁹ is —(CH₂)_(m)—W where m is        from 2, 3, 4 or 5 and W is —OH or halogen (F, Cl, Br or I); and    -   (b) a second species selected from the group consisting of        pharmaceutically acceptable metal Ions, said metal ions having a        valency of n, lysine, arginine and aminosugars,        wherein the formulation has an observed stoichiometry of first        to second species essentially consistent with a notional        stoichiometry of 1:1 except where the second species is a metal        ion having a valency of greater than 1, in which case the        observed stoichiometry is essentially consistent with a notional        stoichiometry of n:1.        128. The formulation of paragraph 127 which has the        characteristic that, after the formulation if not in an aqueous        carrier is placed in one, it has a Ki for thrombin of about 20        nM or less.        129. The formulation of paragraph 127 or 128 in which R⁹ is        3-methoxypropyl and the second species is sodium Ions, lithium        Ions or lysine.        130. The formulation of any of paragraphs 127 to 129 which is in        the form of fine particles for combining with a liquid to form a        liquid formulation        131. A diethanolamine ester of a boronic acid of formula (VIII)

X—(R)-Phe-(S)—Pro-(R)-Mpg-B(OH)₂  (VIII),

where X is H or an amino protecting group.132. A product comprising, in the form of a finely divided solid, a saltconsisting essentially of a monosodium or monolithium salt of an acid ofthe formula Cbz-(R)-Phe-(S)—Pro-(R)-Mpg-H(OH)₂, the salt containing nomore than small amounts of other epimers of said add, the saltoptionally being in admixture with one or more anti-oxidants,preservatives or other additives.133. The product of paragraph 132 in which the salt is in unit dosageform.134. The product of paragraph 132 wherein the unit dosage form is smallvolume parenteral form for injection as an aqueous solution afterreconstitution or is large volume parenteral form for infusion as anaqueous solution after reconstitution.135. The product of any of paragraphs 132 to 134 which further includesan isotonicity agent.136. The product of paragraph 132 which further includes water,optionally having dissolved therein one or more isotonicity agentsand/or other additives, the water being in an amount suitable fordissolving said salt to form a liquid unit dosage form.137. A product comprising, in the form of an Isotonic aqueous solution,a salt consisting essentially of a monosodium or monolithium salt of anacid of the formula Cbz-(R)-Phe-(S)—Pro-(R)-Mpg-B(OH)₂, the saltcontaining no more than small amounts of other epimers of said acid, theproduct optionally further containing one or more anti-oxidants,preservatives or other additives.138. The product of paragraph 137 which is in unit dosage form foradministration by injection or infusion.139. A method of presenting an acid of the formulaCbz-(R)-Phe-(S)—Pro-(R)-Mpg-B(OH)₂ in stabilised form for pharmaceuticaluse, comprising providing the acid in the form of a monosodium,monolithium or monolysine salt thereof and for administration afterreconstitution as an aqueous parenteral solution.

1. A parenteral pharmaceutical formulation comprising a therapeuticallyeffective amount of a pharmaceutically acceptable base addition salt ofa boronic acid of formula (I):

wherein Y comprises a hydrophobic moiety which, together with theaminoboronic acid residue —NHCH(R⁹)—B(OH)₂, has affinity for thesubstrate binding site of thrombin; and R⁹ is a straight chain alkylgroup interrupted by one or more ether linkages and in which the totalnumber of oxygen and carbon atoms is 3, 4, 5 or 6 or R⁹ is —(CH₂)_(m)—Wwhere m is from 2, 3, 4 or 5 and W is —H or halogen.
 2. The formulationof claim 1 wherein R⁹ is an alkoxyalkyl group.
 3. The formulation ofclaim 1 wherein YCO— comprises an amino acid residue which binds to the52 subsite of thrombin, the amino acid residue being N-terminally linkedto a moiety which binds the 53 subsite of thrombin.
 4. The formulationof claim 1 wherein Y comprises a dipeptide which binds to the S3 and S2binding sites of thrombin.
 5. The formulation of claim 4 wherein the53-binding amino acid residue is of (R)-configuration, the 52-bindingresidue Is of (S)-configuration, and the fragment —NHCH(R⁹)—B(OH) is of(R)-configuration.
 6. The formulation of claim 1 wherein R⁹ is analkoxyalkyl group.
 7. The formulation of claim 1 wherein the boronicacid has a Ki for thrombin of about 100 nM or less.
 8. The formulationof claim 1 wherein the salt comprises a salt of the boronic acid withmetal or a strongly basic organic nitrogen-containing compound.
 9. Theformulation of claim 1 wherein the salt comprises a salt of the boronicacid with an alkali metal, an aminosugar, a guanidine or an amine offormula (XI):

where n is from 1 to 6, R² is H, carboxylate or derivatised carboxylate,R³ is H, C₁-C₄ alkyl or a residue of a natural or unnatural amino acid.10. The formulation of claim 4 wherein the Y dipeptide is N-terminallyprotected or N-terminally unprotected, and the peptide linkages in thedipeptide are unsubstituted or independently N-substituted by a C₁-C₁₃hydrocarbyl, wherein the C₁-C₁₃ hydrocarbyl contains no heteratoms or atleast one in-chain or in-ring nitrogen, oxygen or sulfur atom, and theC₁-C₁₃ hydrocarbyl is unsubstituted or substituted by a substituentselected from halo, hydroxy and trifluoromethyl.
 11. The formulation ofclaim 1 wherein the salt consists essentially of an acid salt in whichone B—OH group of formula (I), when trigonally represented, remainsprotonated.
 12. The formulation of claim 9 wherein the salt comprisesboronate ions derived from the peptide boronic acid and has astoichiometry consistent with the boronate ions carrying a singlenegative charge.
 13. The formulation of claim 6 wherein the saltconsists essentially of a monosodium or monolithium salt of the boronicacid.
 14. The pharmaceutical formulation of claim 9 which is adapted forintravenous administration.
 15. A formulation in parenteral dosage formcomprising a therapeutically effective amount of a pharmaceuticallyacceptable base addition salt of a boronic acid of formula (II):

where: X is H or an amino-protecting group; aa¹ is an amino acid residuehaving a hydrocarbyl side chain containing no more than 20 carbon atomsand comprising at least one cyclic group having up to 13 carbon atoms;aa² is an imino acid residue having from 4 to 6 ring members; R¹ is agroup of the formula —(CH₂)_(s)-Z, where s is 2, 3 or 4 and Z is —OH,—OMe, —OEt or halogen.
 16. The formulation of claim 15 wherein aa¹ isselected from Phe, Dpa and wholly or partially hydrogenated analoguesthereof.
 17. The formulation of claim 16 wherein aa¹ is ofR-configuration.
 18. The formulation of claim 15 wherein aa² is aresidue of an imino add of formula (IV)

where R¹ is —CH₂—, —CH₂—CH₂—, —S—CH₂—, —S—C(CH₃)₂— or —CH₂—CH₂—CH₂—,and, when the formula (IV) ring is 5- or 6-membered, the formula (IV)ring is unsubstituted or is substituted at one or more —CH₂— groups byfrom 1 to 3 C₁-C₃ alkyl groups.
 19. The formulation of claim 18 whereinaa² is of S-configuration.
 20. The formulation of claim 15, whereinaa¹-aa² is (R)-Phe-(S)—Pro and the fragment —NH—CH(R¹)—B(OH)₂ is ofR-configuration.
 21. The formulation of claim 16 wherein the boronicacid Is of formula (VIII):X—(R)-Phe-(S)—Pro-(R)-Mpg-B(OH)₂  (VIII), wherein X is R⁶—(CH₂)P—C(O)—,R⁶—(CH₂)_(p)—S(O)₂—, R⁶—(CH₂)_(p)—NH—C(O)— orR⁶—(CH₂)_(p)—O—C(O)-wherein p is 0, 1, 2, 3, 4, 5 or 6 and R⁶ is H or a5 to 13-membered cyclic group which is unsubstituted or substituted by1, 2 or 3 substituents selected from halogen; amino; nitro; hydroxy; aC₅-C₆ cyclic group; C₁-C₄ alkyl and C₁-C₄ alkyl containing, or linked tothe cyclic group through, an in-chain O atom, the aforesaid alkyl groupsoptionally being substituted by a substituent selected from halogen,amino, nitro, hydroxy and a C₅-C₆ cyclic group.
 22. The formulation ofclaim 16 wherein the salt comprises a salt of the boronic acid with analkali metal, an aminosugar or an amine of formula (XI):

where n is from 1 to 6, R² is H, carboxylate or derivatised carboxylate,R³ is H, C₁-C₄ alkyl or a residue of a natural or unnatural amino acid.23. A pharmaceutical product comprising a sealed container containing inthe form of a finely divided solid, ready for reconstitution to form aliquid parenteral formulation, a therapeutically effective amount of aboronate salt which consists essentially of a single pharmaceuticallyacceptable base addition salt of a boronic add formula (II):

where: X is H or an amino-protecting group; aa¹ is an amino acid residueof R-configuration having a hydrocarbyl side chain containing no morethan 20 carbon atoms and comprising at least one cyclic group having upto 13 carbon atoms; aa² is an imino acid residue of S-configurationhaving from 4 to 6 ring members; C* is a chiral centre ofR-configuration; and R¹ is a group of the formula —(CH₂)_(s)-Z, where sis 2, 3 or 4 and Z is —OH, —OMe, —OEt or halogen.
 24. A pharmaceuticalformulation adapted for parenteral administration, whether directly orafter combining with a liquid, and comprising a) a first speciesselected from a boronic acid of formula (I), and boronate ions of saidboronic acid and equilibrium forms of said boronic acid and saidboronate ions:

wherein Y comprises a hydrophobic moiety which, together with theaminoboronic acid residue —NHCH(R⁹)—B(OH)₂, has affinity for thesubstrate binding site of thrombin; and R⁹ is a straight chain alkylgroup interrupted by one or more ether linkages and in which the totalnumber of oxygen and carbon atoms is 3, 4, 5 or 6 or R⁹ is —(CH₂)_(m)—Wwhere m is from 2, 3, 4 or 5 and W is —OH or halogen; and (b) a secondspecies selected from pharmaceutically acceptable metal ions, said metalions having a valency of n, lysine, arginine and aminosugars, whereinthe formulation has an observed stoichiometry of first to second speciesessentially consistent with a notional stoichiometry of 1:1 when thesecond species is a metal ion with a valency of 1 or is lysine, arginineor an aminosugar, or an observed stoichiometry of n:1 when the secondspecies is a metal ion with a valency of greater than
 1. 25. A method ofinhibiting thrombin in the prophylaxis or therapy of disease, comprisingparenterally administering to a mammal suffering from, or at risk ofsuffering from, thrombosis a therapeutically effective amount of thesalt defined in claim
 1. 26. A method for preventing thrombosis in ahaemodialysis circuit of a patient, for preventing a cardiovascularevent In a patient with end stage renal disease, for preventing venousthromboembolic events in a patient receiving chemotherapy through anindwelling catheter, for preventing thromboembolic events in a patientundergoing a lower limb arterial reconstructive procedure, or fortreating by way of therapy or prophylaxis an arterial disease selectedfrom acute coronary syndromes, cerebrovascular thrombosis, peripheralarterial occlusion and arterial thrombosis resulting from atrialfibrillation, valvular heart disease, arterio-venous shunts, indwellingcatheters or coronary stents, the method comprising parenterallyadministering to a mammal a therapeutically effective amount of the saltdefined in claim
 16. 27. A method for making a salt of claim 1,comprising: combining in a solvent diethanolamine and an ester of aboronic acid as defined in claim 1; allowing or causing a precipitate toform and recovering the precipitate; converting the precipitatedmaterial into the free organoboronic acid by contacting the precipitatedmaterial with an aqueous acid or base; and reacting the organoboronicacid with a base of a pharmaceutically acceptable multivalent metal toform to a salt as defined in claim
 1. 28. A medicament adapted forparenteral administration and comprising a therapeutically effectiveamount of a pharmaceutically acceptable base addition salt of a boronicacid which is a selective thrombin Inhibitor and has a neutralaminoboronic acid residue capable of binding to the thrombin S1 subsitelinked through a peptide linkage to a hydrophobic moiety capable ofbinding to the thrombin S2 and 53 subsites, the salt comprising a cationhaving a valency n and having an observed stoichiometry consistent witha notional stoichiometry (boronic acid:cation) of n:
 1. 29. A medicamentof claim 28 wherein the boronic acid has a Ki for thrombin of about 100nM or less.